Preparation method of zinc-dihydromyricetin nano-enzyme and application of zinc-dihydromyricetin nano-enzyme in preparation of drugs for promoting healing of diabetic wounds

Abstract

The invention discloses a preparation method of a zinc-dihydromyricetin nano-enzyme and application of the zinc-dihydromyricetin nano-enzyme in preparation of drugs for promoting healing of diabetic wounds, and the zinc-dihydromyricetin metal polyphenol nano-enzyme (Zn-DHM) is obtained by coordinating a metal element zinc (Zn) and a natural product dihydromyricetin (DHM) through a one-step method by taking PVP as a surfactant. The Zn-DHM nano-enzyme prepared by the invention has antioxidant property and cell proliferation and migration promoting ability, and can maintain metabolism and immune homeostasis of diabetic wounds, so that the Zn-DHM nano-enzyme can accelerate early healing of the diabetic wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of inorganic nanomaterial preparation, and particularly relates to a method for preparing a zinc-dihydromyricetin nanozyme which has antioxidant properties and the ability to maintain metabolic and immune homeostasis and can promote early healing of diabetic wounds. Background Art

[0002] Diabetes is a common chronic metabolic disease in clinical practice. Long-term hyperglycemia can easily lead to various serious complications, one of which is difficult-to-heal wounds, which affect about 25% of diabetic patients. In severe cases, amputation may occur, affecting the patient's health and quality of life. Under normal blood sugar, when the skin barrier is damaged, the body will initiate a wound healing cascade reaction, stimulate the release of multiple growth factors, promote angiogenesis and the synthesis of extracellular matrix such as collagen to achieve wound tissue regeneration, and often go through four stages: hemostasis, inflammation, proliferation, and remodeling. However, diabetic wounds have varying degrees of cellular dysfunction and cannot complete wound structure and function repair within a specific time at each stage like normal blood sugar wounds. Previous studies on the treatment of diabetic wounds have mainly focused on destroying the ROS-inflammatory cascade cycle. Although certain therapeutic effects have been achieved, they have not achieved ideal results.

[0003] The latest research has found that there are serious immune disorders and cell metabolism disorders in early diabetic wounds. In addition to macrophages, Naive CD4+T Cells also play an important role in wound healing. Under long-term hyperglycemia stimulation, various cytokines will cause Naive CD4+T Cells to differentiate more toward Th17 and less toward Treg, thereby increasing the release of IL-17 proinflammatory factors, overactivating the IL-17 signaling pathway, and amplifying inflammation; FOXP3, as the main transcription factor of Treg, plays an important role in maintaining the balance of the immune system and inhibiting inappropriate immune responses. Reduced Treg differentiation leads to immune disorders. In addition, when HUVEC cells addicted to glycolysis metabolism are in a high-sugar environment in the early stage, the increase in intracellular blood glucose content will lead to excessive glycolysis, and excessive intermediates will lead to increased bypass metabolism such as polyols and methylglyoxal, causing increased production of byproducts such as AGEs, ROS, and lactic acid, leading to endothelial dysfunction and tissue acidification, thereby slowing wound healing. Therefore, there is an urgent need to develop a therapeutic strategy that can both resist oxidation and regulate metabolic and immune homeostasis, thereby downregulating the level of inflammation to enable it to smoothly enter the proliferation and remodeling phase and achieve early healing.

[0004] Since it was reported in 2007, nanozymes have been found to have enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). When used in diabetic wounds, they can remove excess ROS and restore the homeostasis of the internal environment. Metal polyphenol nanozymes prepared by combining metal nanomaterials with natural antioxidant products can not only significantly improve antioxidant and anti-inflammatory capabilities, but also have the characteristics of easy preparation, easy storage, and high biocompatibility, and can have the original functions of metal ions and compounds. Zinc is a trace element necessary for human metabolism and growth. As a component of many enzymes, it plays an important role in various physiological processes. Exogenous Zn 2+ The introduction of Zn can restore the activity of Cu / Zn SOD to remove ROS. 2+ It can also inhibit the activation of signaling pathways such as NF-κB and IL-17 and inflammatory cytokines. 2+ It is also involved 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 the woody vine of the genus Ampelopsis or jujube of the grape family. In addition to having anti-inflammatory and antioxidant functions, it can also control blood lipids and blood sugar by regulating cell metabolism. The present invention found that the functional groups such as hydroxyl and carbonyl in DHM contain oxygen atoms, which have strong coordination ability and can bind to Zn 2+ The formation of coordination bonds, and the super delocalization and large π bond conjugation system of DHM make the electron cloud distribution in the dihydromyricetin molecule more uniform, which can 2+ Therefore, Zn 2+ The metal polyphenol nanozyme Zn-DHM coordinated with DHM has the functions of two ligands, which not only has antioxidant ability, but also can regulate metabolism and immune homeostasis, accelerating the healing of early diabetic wounds. A variety of metal polyphenol nanozymes with antioxidant ability have been widely used in the field of human health care, but the application of Zn-DHM nanozyme with the ability to regulate metabolism and immune homeostasis in biomedicine has not been reported so far. 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 preparing a drug for promoting diabetic wound healing.

[0006] In order to achieve the purpose, the present invention adopts the following technical scheme:

[0007] The present invention first discloses a method for preparing a zinc-dihydromyricetin nanozyme, which is characterized in that: ZnCl2 solid and dihydromyricetin DHM powder are used as raw materials, PVP is used as a surfactant, and a zinc-dihydromyricetin metal polyphenol nanozyme is prepared by stirring and reacting, which is recorded as Zn-DHM nanozyme. The specific steps are:

[0008] Dissolving PVP in a solvent to obtain a PVP solution; dissolving ZnCl2 solid in a solvent by ultrasonication to obtain a ZnCl2 solution; dissolving DHM in a solvent to obtain a DHM solution;

[0009] The ZnCl2 solution and the PVP solution were mixed and stirred evenly, and then the DHM solution was added dropwise while stirring. After the addition was completed, the stirring reaction was continued for 24 to 48 hours. The resulting reaction solution was dialyzed to obtain the Zn-DHM nanozyme solution, and then freeze-dried to obtain the 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 consists of methanol and N,N-dimethylformamide (DMF) in a volume ratio of 1:1.

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

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

[0014] The invention also discloses the application of Zn-DHM nanozyme in preparing a medicine for promoting diabetic wound healing.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1. The present invention prepares Zn-DHM nanozyme with good dispersibility, the method is simple and the product morphology is uniform.

[0017] 2. The Zn-DHM nanozyme prepared by the present invention has excellent antioxidant properties and SOD-like enzyme activity, can effectively destroy the ROS-inflammatory cascade cycle, and can promote cell proliferation and migration. In addition, Zn-DHM nanozyme can regulate sugar metabolism, reduce the glucose uptake of HUVEC cells in a high sugar environment, lower the excessive level of intracellular glucose, inhibit the generation of lactic acid, a product of excessive glycolysis, and restore cell function. At the same time, Zn-DHM nanozyme can also regulate immune homeostasis. On the one hand, it can lower the M1 / M2 level, reduce the release of inflammatory factors and promote tissue repair. On the other hand, it can maintain the Th17 / Treg level steady state, reduce the differentiation of Naive CD4+T Cell to Th17, and upregulate the differentiation of Naive CD4+T Cell to Treg, and maintain immune homeostasis. Therefore, the Zn-DHM nanozyme of the present invention has a wide range of application prospects in the preparation of drugs that promote diabetic wound healing.

[0018] 3. In the process of promoting diabetic wound healing, the Zn-DHM nanozyme prepared by the present invention reduces apoptosis by removing ROS and maintaining the mitochondrial membrane potential level on the one hand, and maintains immune and metabolic homeostasis to restore cell function on the other hand. Therefore, a lower dose of Zn-DHM nanozyme can be used to promote early healing of diabetic wounds, and it has higher biosafety.

[0019] 4. When preparing Zn-DHM nanozyme, the present invention uses a mixed solution of methanol and N,N-dimethylformamide as a solvent. Compared with using pure water as a solvent, it is beneficial to the coordination of Zn ions and DHM, and can make the Zn ion content in the product higher. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 This is the XRD image of the Zn-DHM nanozyme prepared in Example 1.

[0022] Figure 3 This is the XPS image 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 elements, and C is the fine spectrum of oxygen elements.

[0023] Figure 4 This is the FTIR image of the Zn-DHM nanozyme prepared in Example 1.

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

[0025] Figure 6 The Zn-DHM nanozyme prepared in Example 1 promotes HUVEC cell proliferation, migration and angiogenesis in vitro, wherein: A is the detection of HUVEC cell activity under treatment with different concentrations of Zn-DHM nanozyme; B is the Zn-DHM nanozyme-induced HUVEC cell migration experiment; C is the Zn-DHM nanozyme-induced HUVEC blood vessel formation experiment.

[0026] Figure 7 These are the damage inhibition test results of the Zn-DHM nanozyme prepared in Example 1 after treating HUVEC cells in a high sugar environment, wherein: A is the ROS clearance of HUVEC cells treated with different concentrations of Zn-DHM nanozyme; B is the mitochondrial membrane potential level of HUVEC cells treated with different concentrations of Zn-DHM nanozyme; C is the apoptosis of HUVEC cells treated with different concentrations of Zn-DHM nanozyme.

[0027] Figure 8 These are the metabolic homeostasis detection results of HUVEC cells treated with Zn-DHM nanozyme prepared in Example 1 under high sugar environment, wherein: A is the glycolysis level of HUVEC cells treated with different concentrations of Zn-DHM nanozyme; B is the glycolytic reserve, which reflects the ability of cells to meet energy needs; C is the glucose uptake level of HUVEC cells treated with different concentrations of Zn-DHM nanozyme; D is the lactate level of HUVEC cells treated with different concentrations of Zn-DHM nanozyme.

[0028] Fig. 9 These are the experimental results of maintaining horizontal homeostasis of immune cells in a high-sugar environment under the treatment of Zn-DHM nanozyme prepared in Example 1, wherein: A is the fluorescence staining image of macrophages polarized to M1 / M2 level under treatment with different concentrations of Zn-DHM nanozyme; B is the flow cytometry result of macrophages polarized to M1 / M2 level under treatment with different concentrations of Zn-DHM nanozyme; C is the ELISA detection result of Naive CD4+T Cell polarization to Th17 under treatment with different concentrations of Zn-DHM nanozyme; D is the ELISA detection result of Naive CD4+T Cell polarization to Treg under treatment with different concentrations of Zn-DHM nanozyme.

[0029] Fig.10 This is a picture of the early healing of wounds in diabetic mice treated with the Zn-DHM nanozyme prepared in Example 1. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail in combination with the embodiments below. The following content is only an example and explanation of the concept of the present invention. The technicians in the relevant technical field make various modifications or supplements to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

[0031] Example 1

[0032] In this example, Zn-DHM nanozyme was prepared 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 volume ratio of 1:1) to obtain a PVP solution. Dissolve 100 mg of ZnCl2 solid in 5 mL of MD solvent by ultrasound to obtain a ZnCl2 solution. Dissolve 50 mg of DHM in 5 mL of MD solvent to obtain a DHM solution.

[0034] The ZnCl2 solution and the PVP solution were mixed and stirred evenly, and then the DHM solution was added dropwise while stirring. After the addition was completed, the stirring reaction continued for 24 hours. The solution after the reaction was placed in a dialysis bag with a molecular weight of 8000-14000KD and dialyzed for 24 hours. During the dialysis process, the deionized water was replaced every 8 hours to obtain the Zn-DHM nanozyme solution. After solidification in a -80°C refrigerator overnight, it was placed in a -60°C vacuum freeze dryer for 24 hours to obtain the Zn-DHM nanozyme powder. The ICP test results showed that the Zn ion content in the product was 10μg / mg.

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

[0036] 1. Morphology characterization

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

[0038] Figure 2 This is the XRD image of the Zn-DHM nanozyme prepared in Example 1. The results show that there is Zn element in Zn-DHM, which has a non-crystalline structure.

[0039] Figure 3The XPS images of the Zn-DHM nanozyme prepared in Example 1 are shown in Figure A. The results show that Zn-DHM is composed of Zn, C, N, O and other elements, which are located at 976.08eV Zn 2p, 531.08eV O1s, 399.08eV N1s and 285.08eV C1s. The peaks of each element are further analyzed, as shown in Figure B. The peaks of Zn 2p at 1044.48eV and 1021.58eV are respectively Zn 2+ 2p 1 / 2 and Zn 2+ 2p 3 / 2 As shown in Figure C, the peak of O 1s at 532.58 eV is -OH, and the peaks at 531.48 eV and 530.48 eV are both MO. These results show that the valence of Zn ions in Zn-DHM is +2, further verifying that Zn 2+ Successfully coordinated with DHM.

[0040] Figure 4 The FTIR image of the Zn-DHM nanozyme prepared in Example 1 shows that the vibration frequency (1050 cm- 1 ~1250cm -1 ) remained basically unchanged, indicating that the C ring ether bond did not open, and the complex -1 The new absorption peak at can be considered to be the stretching vibration and bending vibration absorption of MO, that is, Zn 2+ Zn-DHM is synthesized by coordinating Zn-O bond with DHM.

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

[0042] Figure 5 The results of the in vitro antioxidant properties and enzyme-like activity test of the Zn-DHM nanozyme prepared in Example 1 are shown in the test method according 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 is a DPPH scavenging probe experiment. With the increase of Zn-DHM concentration (quantified by Zn ion concentration), the color of the test solution gradually changed from dark purple to light purple and light yellow within 5 minutes, and its absorption peak at 517nm also decreased continuously. When the Zn-DHM concentration was 1μg / mL, OD 517 It dropped from the initial 0.60au to 0.07au.

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

[0045] C is the detection of SOD enzyme activity. With the increase of Zn-DHM concentration (quantified by Zn ion concentration), its absorption peak at 450nm also decreased continuously. When the Zn-DHM concentration was 10 μg / mL, the clearance rate almost reached 63.33%, indicating that Zn-DHM has good SOD-like enzyme activity.

[0046] 3. Test of HUVEC cell proliferation, migration and angiogenesis

[0047] Figure 6 These are the test results of the Zn-DHM nanozyme prepared in Example 1 in promoting HUVEC cell proliferation, migration and angiogenesis in vitro.

[0048] CCK8 experiment: First, the CCK8 experiment was performed to detect the activity of HUVEC 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 HUVEC, and the effect was best at a concentration of 20 μg / mL. When the Zn-DHM concentration was greater than 50 μg / mL, it would inhibit the activity of the cells.

[0049] Transwell experiment: Since there are nutrients such as serum in the bottom chamber of the Transwell chamber, HUVEC will be induced to migrate from the top chamber to the bottom chamber. After adding different materials of the same concentration to the bottom chamber (Zn-DHM concentration quantified by Zn ion concentration is 20μg / mL, ZnCl2 concentration quantified by Zn ion concentration is 20μg / mL, DHM concentration is 100μg / mL), the results are shown in Figure B. At 6h, compared with the Control group, ZnCl2, DHM, and Zn-DHM groups can promote HUVEC cell migration, and the Zn-DHM group is significantly more than the ZnCl2 and DHM groups.

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

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

[0052] 4. High glucose damage HUVEC cell model

[0053] In this study, a high-glucose (35mM) damaged HUVEC cell model was established to investigate the inhibitory effect of different doses of Zn-DHM nanozymes on the damage of HUVEC cells in a high-glucose environment. The culture time was 48h. The Zn ion concentration was quantified. The Zn-DHM concentration in the high-dose group (High) was 20μg / mL, and the Zn-DHM concentration in the low-dose group (Low) was 10μg / mL.

[0054] like Figure 7 As shown in A, ROS staining fluorescence showed 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 the cells, it can be observed that the intracellular ROS is significantly reduced, and it has a certain concentration dependence.

[0055] When HUVEC is in a high-glucose environment, the excessive production of ROS will lead to the opening of the mitochondrial permeability transition pore (MPTP), and the positive ions or protons in the membrane gap will enter the matrix, causing the ion gradient on both sides of the inner membrane to disappear, thereby affecting the function of mitochondria. Figure 7 As shown in B, the JC-1 staining experiment showed that when HUVEC cells were treated with HG for 48 h, the mitochondrial membrane potential of the cells became low, and JC-1 staining existed in monomeric form, emitting green fluorescence. However, after Zn-DHM treatment, the mitochondrial membrane potential of the cells maintained a certain level, and JC-1 formed J-aggregates, emitting red fluorescence, confirming that Zn-DHM protected the mitochondrial membrane potential level.

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

[0057] 5. Glycolysis level test

[0058] To explore whether Zn-DHM can regulate glucose metabolism disorders, this study first established a high-glucose (35mM) damaged HUVEC cell model, and added different doses of Zn-DHM nanozymes to the model for treatment. The culture time was 48h, among which the Zn ion concentration was quantified. The Zn-DHM concentration in the high-dose group (High) was 20μg / mL, and the Zn-DHM concentration in the low-dose group (Low) was 10μg / mL.

[0059] Seahorse was used to detect the extracellular acidification rate to assess the glycolysis level of cells. Figure 8 As shown in A, after the first addition of saturated glucose, the Glycolysis (basal glycolysis level) of cells increased significantly after 48 hours of HG treatment compared to the Control group, while the Glycolysis (basal glycolysis level) of cells in the Zn-DHM treatment group decreased significantly with the increase of its concentration. The second drug added was oligomycin, which is an inhibitor of ATP synthase and can inhibit mitochondrial ATP production, thereby transferring energy production to the glycolysis pathway. The maximum ECAR value reached by the cell is the glycolytic capacity. The results show that compared with the Control group, the HG treatment group has the highest glycolytic capacity, and the glycolytic capacity decreases after Zn-DHM treatment. The third addition of 2-DG inhibits glycolysis by competitively binding to hexokinase in the glycolysis pathway, causing a decrease in ECAR, thus confirming that the generation of ECAR in the experiment comes from the glycolysis pathway. The difference between Glycolytic capacity and Glycolysis is the Glycolytic reserve, which reflects the ability of cells to meet energy needs. The results in Figure 8B show that the cells' energy needs increased significantly after HG treatment, while there was no significant difference between the low and high concentrations of Zn-DHM treatment and the HG group, proving that Zn-DHM does not reduce cell energy needs. These results show that Zn-DHM can reduce excessive glycolysis caused by HG, thereby reducing the production of the final product lactic acid.

[0060] To investigate whether Zn-DHM can maintain the intracellular glucose homeostasis, glucose uptake ( Figure 8 C) and lactate level test ( Figure 8 D), the results showed that after HG treatment of HUVEC, the glucose uptake level was significantly increased, accompanied by increased lactate production, while in the Zn-DHM treatment group, as its concentration increased, the glucose uptake decreased, causing the intracellular glucose level to approach a steady state, thereby reducing lactate release. These results prove that Zn-DHM can reduce excessive glucose uptake and maintain a steady state of intracellular glucose levels.

[0061] 6. Immune regulation ability detection

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

[0063] After the model was established, CD86 / 206 fluorescence staining was used to mark M1 / M2. Fig. 9 As shown in A, compared with the Control group, after 48 h of HG treatment, the polarization of macrophages toward M1 increased and that toward M2 decreased. However, after Zn-DHM treatment, the polarization of macrophages toward M1 gradually decreased and that toward M2 increased, and this was concentration-dependent. The flow cytometry results also verified that Zn-DHM has the ability to regulate macrophage polarization ( Fig. 9 B).

[0064] In order to explore whether Zn-DHM nanozyme can affect the polarization level of Naive CD4+T Cells to Th17 / Treg, the spleen of mice was first extracted and ground, and Naive CD4+T Cells were extracted by magnetic beads, and then amplified and activated using CD3 / 28 antibodies to obtain cells with the ability to polarize to Th17 and Treg. Then HG, IL-6, TGF-β and IFN-γ were added to the culture medium to establish a Naive CD4+T Cell polarization group to Th17, and Zn-DHM was added to observe its ability to regulate the polarization of Naive CD4+T Cells to Th17 / Treg. Fig. 9 As shown in C and D, compared with the Control group, the secretion of IL-17 in the polarized group was significantly higher, while FOXP3 was significantly lower. 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. After one week of adaptive feeding, they were fed with a high-sugar and high-fat diet for 6 weeks and continuously intraperitoneally injected with STZ solution for 5 days. The diabetic mouse model was successfully established when the fasting blood glucose of the mice was ≥11.1mmo / L. The diabetic mice with successful modeling were randomly divided into 4 groups, with 5 mice in each group, namely PBS group (Control), ZnCl2, DHM group, and Zn-DHM treatment group (Zn-DHM group). The back was cut with a wound of about 1cm in diameter, and 200μL of drug was dripped into each group for treatment. The drug was administered once a day for the first three days. ,

[0067] Fig.10 This is a picture of the early healing of wounds in diabetic mice treated with Zn-DHM nanozyme prepared in Example 1. The photographic records of the mouse wounds showed that the wounds in the Control group healed 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. On the 14th day, the wounds were almost completely healed, and new hair appeared on the surface of the new skin.

[0068] The above description is only 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 in the protection scope of the present invention.

Claims

1. A method for preparing zinc-dihydromyricetin nanozyme, characterized in that: Using ZnCl2 solid and dihydromyricetin DHM powder as raw materials and PVP as surfactant, zinc-dihydromyricetin nanozyme was prepared by stirring reaction, which was recorded as Zn-DHM nanozyme.

2. The method for preparing the zinc-dihydromyricetin nanozyme according to claim 1, characterized in that: Dissolving PVP in a solvent to obtain a PVP solution; dissolving ZnCl2 solid in a solvent to obtain a ZnCl2 solution; dissolving DHM in a solvent to obtain a DHM solution; The ZnCl2 solution and the PVP solution were mixed and stirred evenly, and then the DHM solution was added dropwise while stirring. After the addition was completed, the stirring reaction was continued for 24 to 48 hours. The resulting reaction solution was dialyzed to obtain the Zn-DHM nanozyme solution, and then freeze-dried to obtain the Zn-DHM nanozyme powder.

3. The method for preparing the zinc-dihydromyricetin nanozyme according to claim 1 or 2, characterized in that: The mass ratio of ZnCl2, DHM and PVP is 1.5-2.5:1:5-10.

4. The method for preparing the zinc-dihydromyricetin nanozyme according to claim 2, characterized in that: The solvent is composed of methanol and N,N-dimethylformamide in a volume ratio of 1:

1.

5. The method for preparing the zinc-dihydromyricetin nanozyme according to claim 2, characterized in that: The molecular weight of the dialysis bag used for the dialysis is 8000-14000 KD, the dialysis time is 24-48 hours, and the deionized water is replaced every 8 hours.

6. A zinc-dihydromyricetin nanozyme prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the zinc-dihydromyricetin nanozyme according to claim 6 in the preparation of a drug for promoting diabetic wound healing.

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