Preparation method of zinc-dihydromyricetin nanoscale enzyme and application thereof in preparation of medicine for promoting healing of diabetic wounds

Abstract

The application discloses a preparation method of zinc-dihydromyricetin nano-enzyme and application of the zinc-dihydromyricetin nano-enzyme in preparation of a drug for promoting wound healing of diabetes, and is characterized in that: a metal element zinc (Zn) is coordinated with a natural product dihydromyricetin (DHM) through a one-step method to obtain zinc-dihydromyricetin metal polyphenol nano-enzyme (Zn-DHM) by taking PVP as a surfactant. The Zn-DHM nano-enzyme prepared in the application has the properties of antioxidation, cell proliferation and migration promotion, and can maintain the metabolic and immune homeostasis of a wound of diabetes, so that the early healing of the wound of diabetes can be accelerated.

Description

Technical Field

[0001] This invention belongs to the field of inorganic nanomaterial preparation technology, and specifically relates to a method for preparing a zinc-dihydromyricetin nanoenzyme with antioxidant properties and the ability to maintain metabolic and immune homeostasis, which can promote the early healing of diabetic wounds. Background Technology

[0002] Diabetes mellitus is a common chronic metabolic disease. Long-term hyperglycemia can easily lead to various serious complications, one of which is non-healing wounds, affecting approximately 25% of diabetic patients. In severe cases, it can lead to amputation, impacting patients' health and quality of life. Under normal blood glucose levels, when the skin barrier is damaged, the body initiates a wound healing cascade, stimulating the release of various growth factors, promoting angiogenesis and the synthesis of extracellular matrix proteins such as collagen to achieve wound tissue regeneration. This process typically involves four stages: hemostasis, inflammation, proliferation, and remodeling. However, diabetic wounds exhibit varying degrees of cellular dysfunction, preventing them from completing structural and functional repair at the specific timeframes of each stage, unlike wounds with normal blood glucose levels. Previous research on treating diabetic wounds has primarily focused on disrupting the ROS-inflammatory cascade cycle, achieving some therapeutic effects but not reaching the ideal outcome.

[0003] Recent research has revealed severe immune dysregulation and cellular metabolic disorders in early diabetic wounds. Besides macrophages, naive CD4+ T cells also play a crucial role in wound healing. Under prolonged hyperglycemic stimulation, various cytokines lead to increased differentiation of naive CD4+ T cells into Th17 cells and decreased differentiation into Treg cells, resulting in increased release of the pro-inflammatory IL-17 factor, overactivation of the IL-17 signaling pathway, and amplification of inflammation. FOXP3, as a major transcription factor for Treg cells, plays a vital role in maintaining immune system balance and suppressing inappropriate immune responses; reduced Treg differentiation leads to immune dysregulation. Furthermore, HUVEC cells addicted to glycolysis experience excessive intracellular glucose levels in the early stages of a high-glucose environment, leading to excessive glycolysis. Excessive intermediate products result in increased bypass metabolism to polyols and methylglyoxal, causing increased production of byproducts such as AGEs, ROS, and lactic acid, leading to endothelial dysfunction and tissue acidification, thus slowing wound healing. Therefore, there is an urgent need to develop a treatment strategy that can both resist oxidation and regulate metabolism and immune homeostasis, thereby downregulating inflammation levels to facilitate the proliferative and remodeling phases and achieve early healing.

[0004] Since its initial report in 2007, nanozymes have been found to possess enzymatic activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Their application in diabetic wounds can remove excess reactive oxygen species (ROS) and restore homeostasis. Metal polyphenol nanozymes prepared by combining metal nanomaterials with natural antioxidants not only significantly enhance antioxidant and anti-inflammatory capabilities but also possess characteristics such as ease of preparation, storage, and high biocompatibility. Furthermore, they retain the original functions of metal ions and compounds. Zinc is an essential trace element for human metabolism and growth, playing a crucial role in various physiological processes as a component of many enzymes. Exogenous Zn... 2+ The introduction of Zn can achieve the effect of scavenging ROS by restoring Cu / Zn SOD activity. 2+ It can also inhibit the activation of signaling pathways such as NF-κB and IL-17, as well as inflammatory cytokines. Zn 2+ It also participates in the wound healing process, such as inducing the release of vascular endothelial growth factor and promoting angiogenesis, regulating the expression of extracellular proteins such as collagen and keratin, and accelerating epidermal cell division and DNA repair. DHM is a flavonoid compound extracted from woody vines of the genus *Viburnum* or *Hovenia dulcis* in the Vitaceae family. In addition to its anti-inflammatory and antioxidant functions, it can also control blood lipids and blood sugar by regulating cell metabolism. The research of this invention found that the hydroxyl and carbonyl functional groups in DHM contain oxygen atoms, which have a strong coordinating ability and can interact with Zn. 2+ The formation of coordinate bonds, coupled with the hyperdelocalization and large π-bond conjugation of DHM, results in a relatively uniform electron cloud distribution within the dihydromyricetin molecule, enabling it to interact with Zn. 2+ It forms stable complexes. Therefore, Zn 2+ The metallopolyphenol nanozyme Zn-DHM, synthesized by coordination with DHM, possesses the functions of both ligands: antioxidant capacity and the ability to regulate metabolism and immune homeostasis, thus accelerating the healing of early diabetic wounds. While various metallopolyphenol nanozymes with antioxidant capabilities have been widely used in human healthcare, the biomedical applications of Zn-DHM nanozymes, which regulate metabolism and immune homeostasis, have not yet been reported. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing zinc-dihydromyricetin nanozyme and its application in the preparation of drugs that promote the healing of diabetic wounds.

[0006] To achieve its objectives, the present invention employs the following technical solution:

[0007] This invention first discloses a method for preparing zinc-dihydromyricetin nanozyme, characterized by: using ZnCl2 solid and dihydromyricetin (DHM) powder as raw materials, and PVP as a surfactant, a stirring reaction is carried out to obtain zinc-dihydromyricetin metal polyphenol nanozyme, denoted as Zn-DHM nanozyme. The specific steps are as follows:

[0008] PVP was dissolved in a solvent to obtain a PVP solution; ZnCl2 solid was ultrasonically dissolved in a solvent to obtain a ZnCl2 solution; DHM was dissolved in a solvent to obtain a DHM solution.

[0009] The ZnCl2 solution and PVP solution were mixed and stirred until homogeneous. Then, DHM solution was added dropwise while stirring. After the addition was completed, the reaction was stirred for 24–48 h. The resulting reaction solution was dialyzed to obtain Zn-DHM nanozyme solution, which was then freeze-dried to obtain Zn-DHM nanozyme powder.

[0010] Preferably, the mass ratio of ZnCl2, DHM and PVP is 1.5-2.5:1:5-10.

[0011] Preferably, the solvent is composed of methanol and N,N-dimethylformamide (DMF) in a volume ratio of 1:1.

[0012] Preferably, the dialysis bag used for dialysis has a molecular weight of 8000-14000KD, the dialysis time is 24-48 hours, and the deionized water is replaced every 8 hours. The purpose of dialysis is to purify the solution.

[0013] The Zn-DHM nanozyme prepared by this invention has good water dispersibility and can be dispersed in liquids such as water, physiological saline, PBS buffer, and cell culture medium, as well as in hydrogels such as sodium alginate hydrogel and F127 hydrogel.

[0014] This invention also discloses the application of Zn-DHM nanozymes in the preparation of drugs that promote the healing of diabetic wounds.

[0015] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0016] 1. The present invention has prepared Zn-DHM nanozymes with good dispersibility. The method is simple and the product has a uniform morphology.

[0017] 2. The Zn-DHM nanozyme prepared in this invention possesses superior antioxidant properties and SOD-like enzyme activity, effectively disrupting the ROS-inflammatory cascade and promoting cell proliferation and migration. Furthermore, the Zn-DHM nanozyme can regulate glucose metabolism, reducing glucose uptake in HUVEC cells under high glucose conditions, downregulating excessively high intracellular glucose levels, inhibiting the production of lactic acid, a product of excessive glycolysis, and restoring cell function. Simultaneously, the Zn-DHM nanozyme can also regulate immune homeostasis. On the one hand, it can downregulate M1 / M2 levels, reduce the release of inflammatory factors, and promote tissue repair; on the other hand, it can maintain Th17 / Treg homeostasis, reduce the differentiation of naive CD4+ T cells into Th17 cells, and upregulate the differentiation of naive CD4+ T cells into Treg cells, thus maintaining immune homeostasis. Therefore, the Zn-DHM nanozyme of this invention has broad application prospects in the preparation of drugs that promote diabetic wound healing.

[0018] 3. The Zn-DHM nanozyme prepared in this invention reduces apoptosis by clearing ROS and maintaining mitochondrial membrane potential levels during the process of promoting the healing of diabetic wounds. On the other hand, it maintains immune and metabolic homeostasis and restores cell function. Therefore, a lower dose of Zn-DHM nanozyme can be used to promote the early healing of diabetic wounds and has high biosafety.

[0019] 4. In the preparation of Zn-DHM nanozymes, the present invention uses a mixture of methanol and N,N-dimethylformamide as a solvent. Compared with using pure water as a solvent, this is more conducive to the coordination of Zn ions with DHM, and can result in a higher Zn ion content in the product. Attached Figure Description

[0020] Figure 1 This is a TEM image of the Zn-DHM nanozyme prepared in Example 1.

[0021] Figure 2 The image shows the XRD pattern of the Zn-DHM nanozyme prepared in Example 1.

[0022] Figure 3 XPS images of the Zn-DHM nanozyme prepared in Example 1, where A is the full spectrum of Zn-DHM, B is the fine spectrum of zinc, and C is the fine spectrum of oxygen.

[0023] Figure 4 The image shows an FTIR image of the Zn-DHM nanozyme prepared in Example 1.

[0024] Figure 5 The following are in vitro assays for the antioxidant properties and enzyme-like activities of the Zn-DHM nanozyme prepared in Example 1, wherein: A is the DPPH·scavenging probe assay; B is the PTIO·scavenging probe assay; and C is the SOD enzyme activity assay.

[0025] Figure 6 The following describes the in vitro proliferative, migratory, and angiogenesis-promoting abilities of the Zn-DHM nanozyme prepared in Example 1, where: A represents the detection of HUVEC cell activity under different concentrations of Zn-DHM nanozyme treatment; B represents the Zn-DHM nanozyme-induced HUVEC cell migration experiment; and C represents the Zn-DHM nanozyme-induced HUVEC angiogenesis experiment.

[0026] Figure 7 The results of the damage inhibition detection of HUVEC cells under high glucose environment by the Zn-DHM nanozyme prepared in Example 1 are shown. Among them: A is the ROS clearance of HUVEC cells under different concentrations of Zn-DHM nanozyme treatment; B is the mitochondrial membrane potential level of HUVEC cells under different concentrations of Zn-DHM nanozyme treatment; C is the apoptosis of HUVEC cells under different concentrations of Zn-DHM nanozyme treatment.

[0027] Figure 8 The results of metabolic homeostasis detection of HUVEC cells under high glucose environment after treatment with Zn-DHM nanozymes prepared in Example 1 are shown below. In this table: A represents the glycolysis level of HUVEC cells under different concentrations of Zn-DHM nanozymes; B represents the glycolytic reserve, which reflects the cell's ability to meet energy requirements; C represents the glucose uptake level of HUVEC cells under different concentrations of Zn-DHM nanozymes; and D represents the lactate level of HUVEC cells under different concentrations of Zn-DHM nanozymes.

[0028] Figure 9 The results of the experiment in Example 1 on the maintenance of homeostasis of immune cells under high glucose environment after treatment with Zn-DHM nanozymes are as follows: A is the fluorescence staining of macrophages polarized to the M1 / M2 level under different concentrations of Zn-DHM nanozymes; B is the flow cytometry result of macrophages polarized to the M1 / M2 level under different concentrations of Zn-DHM nanozymes; C is the ELISA detection result of naive CD4+ T cells polarized to Th17 under different concentrations of Zn-DHM nanozymes; D is the ELISA detection result of naive CD4+ T cells polarized to Treg under different concentrations of Zn-DHM nanozymes.

[0029] Figure 10 The image shows the early wound healing in diabetic mice treated with the Zn-DHM nanozyme prepared in Example 1. Detailed Implementation

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

[0031] Example 1

[0032] This embodiment prepares Zn-DHM nanozymes according to the following steps:

[0033] Weigh 330 mg of polyvinyl pyrrolidone (pvp) and dissolve it in 25 mL of MD solvent (composed of methanol, Methano, and DMF in a 1:1 volume ratio) to obtain a PVP solution. Dissolve 100 mg of solid ZnCl2 completely in 5 mL of MD solvent by sonication to obtain a ZnCl2 solution. Dissolve 50 mg of DHM in 5 mL of MD solvent to obtain a DHM solution.

[0034] ZnCl2 solution and PVP solution were mixed and stirred until homogeneous. Then, DHM solution was added dropwise while stirring. After the addition was complete, the reaction was continued for 24 hours with stirring. The resulting solution was then dialyzed in a dialysis bag with a molecular weight of 8000-14000 KD for 24 hours. During the dialysis process, the deionized water was replaced every 8 hours to obtain a Zn-DHM nanozyme solution. After solidification overnight at -80℃, the solution was freeze-dried in a vacuum freeze dryer at -60℃ for 24 hours to obtain Zn-DHM nanozyme powder. ICP analysis showed that the Zn ion content in the product was 10 μg / mg.

[0035] The Zn-DHM nanozyme obtained in this embodiment was subjected to the following morphological and performance standards:

[0036] 1. Morphological characteristics

[0037] Figure 1 The image shows a TEM image of the Zn-DHM nanozyme prepared in Example 1. It clearly shows the microsphere-like structure of Zn-DHM, which is uniform in structure and extremely small in size, with a diameter of about 5 nm.

[0038] Figure 2 The image shows the XRD pattern of the Zn-DHM nanozyme prepared in Example 1. The results show that Zn-DHM contains Zn elements and has an amorphous structure.

[0039] Figure 3XPS images of the Zn-DHM nanozyme prepared in Example 1 are shown. Image A shows the full spectrum of Zn-DHM, revealing that it is composed of Zn, C, N, and O elements, with peaks at Zn 2p (976.08 eV), O 1s (531.08 eV), N 1s (399.08 eV), and C 1s (285.08 eV), respectively. Further analysis of the spectral peaks for each element is shown in Image B. The Zn 2p peaks at 1044.48 eV and 1021.58 eV represent Zn... 2+ 2p 1 / 2 and Zn 2+ 2p 3 / 2 As shown in Figure C, the O 1s peak at 532.58 eV corresponds to -OH, while the peaks at 531.48 eV and 530.48 eV both correspond to MO. These results indicate that the valence of Zn ions in Zn-DHM is +2, further verifying the Zn ion's valence. 2+ Successful coordination with DHM.

[0040] Figure 4 The image shows an FTIR image of the Zn-DHM nanozyme prepared in Example 1. The results show that the vibrational frequency of the COC bond between the DHM raw material and the Zn-DHM molecule is (1050 cm⁻¹). 1 ~1250cm -1 The structure remained essentially unchanged, indicating that the C-ring ether bond did not undergo ring-opening, and the complex measured 647.4 cm⁻¹. -1 The appearance of a new absorption peak at this point can be attributed to the absorption of stretching and bending vibrations of MO, i.e., Zn. 2+ Zn-DHM was synthesized by coordination with DHM via Zn-O bonds.

[0041] 2. Antioxidant properties and enzyme-like activity detection

[0042] Figure 5 The results of in vitro antioxidant properties and enzyme-like activity assays of the Zn-DHM nanozymes prepared in Example 1 were obtained by referring to the article "Caffeic acid-vanadium nanozymes treat skin flap ischemia-reperfusion injury through macrophage reprogramming and the upregulation of X-linked inhibitors of apoptotic proteins, Acta Pharmaceutica Sinica B, 26 August 2024". Among them:

[0043] A represents the DPPH scavenging probe experiment. With increasing Zn-DHM concentration (quantified by Zn ion concentration), the color of the detection solution gradually changed from dark purple to light purple and then pale yellow within 5 minutes, and its absorption peak at 517 nm continuously decreased. When the Zn-DHM concentration was 1 μg / mL, the OD... 517 It decreased from the initial 0.60au to 0.07au.

[0044] B represents the PTIO· scavenging probe experiment. With increasing Zn-DHM concentration (quantified by Zn ion concentration), the color of the PTIO· test solution gradually changed from blue-purple to light purple within 10 minutes, and its absorption peak at 557 nm continuously decreased. When the Zn-DHM concentration was 10.0 μg / mL, the OD... 557 It decreased from the initial 0.48au to 0.14au.

[0045] C represents the detection of SOD enzyme activity. As the concentration of Zn-DHM (quantified by Zn ion concentration) increases, its absorption peak at 450 nm also decreases continuously. When the concentration of Zn-DHM is 10 μg / mL, the scavenging rate reaches almost 63.33%, indicating that Zn-DHM has good SOD enzyme activity.

[0046] 3. Tests on the ability to promote HUVEC cell proliferation, migration, and angiogenesis.

[0047] Figure 6 The results show the in vitro test results of the Zn-DHM nanozyme prepared in Example 1 on its ability to promote HUVEC cell proliferation, migration and angiogenesis.

[0048] CCK8 assay: First, the activity of HUVECs was detected by setting concentration groups. As shown in Figure A, when the Zn-DHM concentration (quantified by Zn ion concentration) was less than 50 μg / mL, it promoted the proliferation of HUVECs. The effect was best at a concentration of 20 μg / mL. When the Zn-DHM concentration was greater than 50 μg / mL, it inhibited the activity of cells.

[0049] Transwell assay: The presence of serum and other nutrients in the bottom chamber of the Transwell chamber induces HUVECs to migrate from the top chamber to the bottom chamber. The results of adding the same concentrations of different materials (Zn-DHM concentration of 20 μg / mL, ZnCl2 concentration of 20 μg / mL, and DHM concentration of 100 μg / mL) to the bottom chamber are shown in Figure B. At 6 h, compared to the Control group, ZnCl2, DHM, and Zn-DHM groups all promoted HUVEC cell migration, with the Zn-DHM group showing significantly more migration than the ZnCl2 and DHM groups.

[0050] Angiogenesis experiment: The results of the tube formation experiment are shown in Figure C. At 6h, Zn-DHM had a significantly higher ability to promote HUVEC angiogenesis than ZnCl2 and DHM.

[0051] The above experimental results show that Zn-DHM possesses Zn 2+ The properties of Zn-DHM can significantly promote cell proliferation, migration and tube formation, and are superior to those of ZnCl2 and DHM, which means that Zn-DHM will be able to play an important role in the wound healing process.

[0052] 4. High glucose-induced HUVEC cell model

[0053] This study established a high glucose (35 mM) injury model of HUVEC cells and investigated the inhibitory effect of different doses of Zn-DHM nanozymes on HUVEC cells under high glucose conditions. The culture time was 48 h. The results were quantified by Zn ion concentration. The high dose group had a Zn-DHM concentration of 20 μg / mL and the low dose group had a Zn-DHM concentration of 10 μg / mL.

[0054] like Figure 7 As shown in Figure A, ROS staining fluorescence indicates that HG (high glucose 35mM) treatment for 48h can induce the production of a large amount of ROS in HUVEC cells. Since Zn-DHM itself has antioxidant properties and SOD-like enzyme activity, after adding it to the culture medium to treat cells, the intracellular ROS was significantly reduced, and this reduction was in a certain concentration-dependent manner.

[0055] HUVECs in a high-glucose environment experience excessive ROS production, leading to the opening of the mitochondrial permeability transition pore (MPTP). This allows positive ions or protons from the intermembrane space to enter the matrix, causing the ion gradient across the inner membrane to disappear and consequently affecting mitochondrial function. Figure 7 As shown in Figure B, the JC-1 staining experiment showed that after HG treatment of HUVEC cells for 48 hours, the mitochondrial membrane potential of the cells decreased, and JC-1 staining existed in monomeric form, emitting green fluorescence. However, after Zn-DHM treatment, the mitochondrial membrane potential of the cells remained at a certain level, and JC-1 formed J-aggregates, emitting red fluorescence, confirming that Zn-DHM protects the mitochondrial membrane potential level.

[0056] Mitochondrial dysfunction can lead to cell apoptosis, such as... Figure 7 As shown in Figure C, AM / PI staining fluorescence indicated that a large number of dead cells were present in the field of view after HG treatment for 48 hours, while treatment with Zn-DHM showed an increase in live cells and a reduction in apoptosis with increasing concentration.

[0057] 5. Glycolysis level test

[0058] To investigate whether Zn-DHM can regulate glucose metabolism disorders, this study first established a high glucose (35 mM)-induced injury model of HUVEC cells. Different doses of Zn-DHM nanozyme were added to the model for treatment, and the culture time was 48 h. The concentration of Zn ion was quantified as follows: the high dose group (High) had a Zn-DHM concentration of 20 μg / mL, and the low dose group (Low) had a Zn-DHM concentration of 10 μg / mL.

[0059] The Seahorse assay was used to assess cellular glycolysis levels by detecting extracellular acidification. Figure 8 As shown in Figure A, after the first addition of saturated glucose, compared to the Control group, the basal glycolysis level of cells treated with HG was significantly increased after 48 hours. In contrast, the basal glycolysis level of cells treated with Zn-DHM decreased significantly with increasing concentration. The second addition was oligomycin, an inhibitor of ATP synthase, which inhibits mitochondrial ATP production, thereby shifting energy production to the glycolytic pathway. The maximum ECAR value reached by the cells was the glycolytic capacity. The results showed that the HG treatment group had the highest glycolytic capacity compared to the Control group, while the glycolytic capacity decreased after Zn-DHM treatment. The third administration of 2-DG, which inhibits glycolysis by competitively binding to hexokinase in the glycolytic pathway, led to a decrease in ECAR, thus confirming that ECAR production in the experiment originated from the glycolytic pathway. The difference between glycolytic capacity and glycolysis is the glycolytic reserve, reflecting the cell's ability to meet its energy needs. Figure 8B shows that HG treatment significantly increased the cells' ability to meet their energy requirements, while low and high concentrations of Zn-DHM treatment showed no significant difference compared to the HG group, proving that Zn-DHM does not reduce cellular energy requirements. These results indicate that Zn-DHM can reduce excessive glycolysis induced by HG, thereby reducing the production of the final product, lactic acid.

[0060] To investigate whether Zn-DHM can maintain intracellular glucose homeostasis, glucose uptake was studied. Figure 8 C) and lactate level test ( Figure 8 D) The results showed that HG treatment of HUVECs significantly increased glucose uptake, accompanied by increased lactate production. In contrast, the Zn-DHM treatment group showed decreased glucose uptake with increasing concentration, bringing intracellular glucose levels closer to homeostasis and consequently reducing lactate release. These results demonstrate that Zn-DHM can reduce excessive glucose uptake and maintain stable intracellular glucose levels.

[0061] 6. Detection of immune regulation capacity

[0062] To investigate whether Zn-DHM can regulate immune homeostasis, this study first established a high glucose (35 mM) induced inflammatory polarization model of RAW274.7 macrophages. Different doses of Zn-DHM nanozyme were added to the model for treatment, and the culture time was 48 h. The concentration of Zn ion was quantified, with the high dose group (High) having a Zn-DHM concentration of 20 μg / mL and the low dose group (Low) having a Zn-DHM concentration of 10 μg / mL.

[0063] After the model was established, M1 / M2 were labeled with CD86 / 206 fluorescent staining, such as... Figure 9 As shown in Figure A, compared to the Control group, after 48 hours of HG treatment, macrophages showed increased polarization towards M1 and decreased polarization towards M2. However, with Zn-DHM treatment, macrophages gradually showed decreased polarization towards M1 and increased polarization towards M2, exhibiting a concentration-dependent effect. Flow cytometry results also confirmed that Zn-DHM has the ability to regulate macrophage polarization. Figure 9 B).

[0064] To investigate whether Zn-DHM nanozymes can affect the polarization level of naive CD4+ T cells towards Th17 / Treg, mouse spleens were first extracted and ground, and naive CD4+ T cells were extracted using magnetic beads. These cells were then amplified and activated using CD3 / 28 antibodies to obtain cells capable of polarizing into Th17 and Treg cells. HG, IL-6, TGF-β, and IFN-γ were then added to the culture medium to establish a naive CD4+ T cell Th17 polarization group. Simultaneously, Zn-DHM was added to observe its ability to regulate the polarization of naive CD4+ T cells towards Th17 / Treg. Figure 9 As shown in C and D, compared with the Control group, the polarization group secreted significantly higher levels of IL-17 and significantly lower levels of FOXP3. After Zn-DHM treatment, the level of IL-17 was significantly downregulated, while the level of FOXP3 was significantly upregulated.

[0065] 7. Wound healing experiment

[0066] Thirty 5-week-old male C57BL / 6J mice were selected and fed a high-sugar, high-fat diet for 6 weeks after a week of acclimatization. They were then intraperitoneally injected with STZ solution for 5 consecutive days. Successful diabetic mouse modeling was defined as a fasting blood glucose level ≥11.1 mmol / L. The successfully modeled diabetic mice were randomly divided into four groups of five mice each: PBS group (Control), ZnCl2 group, DHM group, and Zn-DHM treatment group (Zn-DHM group). A 1 cm diameter incision was made on the back of each mouse, and 200 μL of the drug was administered to each group once daily for the first three days.

[0067] Figure 10 The images show the early wound healing of diabetic mice treated with Zn-DHM nanozyme prepared in Example 1. The wound photos of the mice show that the wound healing in the Control group was the slowest, with some inflammatory secretions visible on the surface. DHM, ZnCl2 and Zn-DHM all promoted wound healing, but the Zn-DHM group had the best effect. The wound was almost completely healed on the 14th day, and new hair was present on the surface of the new skin.

[0068] The above description is merely an exemplary embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing zinc-dihydromyricetin nanoszyme, characterized in that: Dissolve the surfactant PVP in the solvent to obtain a PVP solution; dissolve ZnCl2 solid in the solvent to obtain a ZnCl2 solution; dissolve dihydromyricetin DHM in the solvent to obtain a DHM solution; mix the ZnCl2 solution and the PVP solution and stir until uniform, then add the DHM solution dropwise while stirring, continue stirring for 24-48 h after the addition is completed, and obtain a Zn-DHM nanoscale enzyme solution by dialysis, and then freeze-dry to obtain a zinc-dihydromyricetin nanoscale enzyme powder, denoted as Zn-DHM nanoscale enzyme; wherein the mass ratio of ZnCl2, DHM and PVP is 1.5-2.5:1:5-10, and the solvent is composed of methanol and N,N-dimethylformamide at a volume ratio of 1:

1.

2. The method for preparing zinc-dihydromyricetin nanoszyme according to claim 1, characterized in that: The dialysis bag used for dialysis has a molecular weight of 8000-14000KD, and the dialysis time is 24-48h, with deionized water being replaced every 8h. 3.A zinc-dihydromyricetin nanoscale enzyme prepared by the preparation method of any one of claims 1-2. 4.Use of the zinc-dihydromyricetin nanoscale enzyme of claim 3 in the preparation of a drug for promoting the healing of a diabetic wound.

Images