Traditional Chinese medicine muscone slow-release nanofiber dressing as well as preparation method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicines, and discloses a traditional Chinese medicine muscone slow-release nanofiber dressing as well as a preparation method and application thereof. The preparation method of the dressing comprises the following steps: respectively dissolving chitosan hydrochloride and pullulan in a glacial acetic acid solution, and mixing the two polysaccharide solutions to form a mixed solution; performing high-voltage electrostatic spinning on the mixed solution to prepare nanofibers, and performing high-temperature crosslinking; completely soaking the obtained nanofibers in a foaming solution, cleaning the foamed nanofibers after stabilization, then carrying out bubble removal treatment, freezing and freeze-drying to obtain porous nanofibers; and dissolving musk ketone in absolute ethyl alcohol, soaking the porous nanofiber in the musk ketone solution, and after the absolute ethyl alcohol is completely volatilized, obtaining the product. The muscone slow-release nanofiber membrane capable of improving blood circulation is prepared from the porous nanofiber loaded with traditional Chinese medicine muscone, loading and slow release of active medicine are facilitated, and the problem that blood circulation is blocked in diabetes wound healing is solved.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and specifically to a sustained-release nano-fiber dressing of muskone in traditional Chinese medicine and its preparation method and application. Background Art

[0002] Diabetic wounds are a common diabetic complication. The wounds are prone to infection and in a state of chronic inflammation, and are difficult to heal, eventually leading to a high amputation rate, bringing a heavy burden to people's lives and society. When the blood glucose level rises, glucose enters pancreatic β cells through glucose transporters, and through a series of actions, voltage-gated calcium channels are activated, resulting in the influx of Ca 2+ ions, thereby causing vasoconstriction, leading to blood circulation disorders, exacerbating the insufficient supply of blood, oxygen and nutrients at the wound site and the occurrence of infections, and ultimately resulting in slow healing of diabetic wounds or even local necrosis and ulcers.

[0003] Traditional treatment methods have inherent limitations in comprehensively dealing with these complex pathological environments, and existing nano-fiber dressings are prone to side effects such as inhibiting angiogenesis due to raw material degradation, etc., thus gradually attracting wide attention. Currently, most of the research on diabetic wound healing focuses on immunomodulation, antibacterial, antioxidant, promoting angiogenesis or even programmatically achieving the above various effects, and there is little research on promoting diabetic wound healing by improving blood circulation. Summary of the Invention

[0004] The purpose of the present invention is to provide a sustained-release nano-fiber dressing of muskone in traditional Chinese medicine and its preparation method and application.

[0005] To achieve the purpose of the present invention, in the first aspect, the present invention provides a preparation method of a sustained-release nano-fiber dressing of muskone in traditional Chinese medicine, including the following steps: S1. Dissolve chitosan hydrochloride and pullulan polysaccharide in acetic acid solution respectively, stir at room temperature (6 - 10 h) until a uniformly dispersed solution is formed to obtain a chitosan hydrochloride solution and a pullulan polysaccharide solution; mix the two polysaccharide solutions in proportion and continue to stir (4 - 8 h), the total polysaccharide concentration in the obtained mixed solution is 10 - 15 wt% (preferably 12.5 wt%), and the mass ratio of chitosan hydrochloride to pullulan polysaccharide is 1:2 - 1:6 (preferably 1:4); S2. Perform high-voltage electrospinning on the mixed solution to prepare nano-fibers, and then crosslink at a high temperature of 100 - 135 °C for 0.5 - 2 h; S3. Completely immerse the nano-fibers obtained in step S2 in a foaming solution, after stabilization, wash the foamed nano-fibers, then perform degassing treatment, freeze and lyophilize to obtain porous nano-fibers; S4. Dissolve muscone in absolute ethanol to obtain a muscone solution; then soak the porous nanofibers in the muscone solution, and wait for the absolute ethanol to completely volatilize to obtain the traditional Chinese medicine muscone sustained-release nanofiber dressing (Musc@CP-NF).

[0006] Further, in step S1, the concentration of the glacial acetic acid solution is 12 - 50% (preferably 50%).

[0007] Further, in step S2, the high-voltage electrospinning control parameters are as follows: the feeding speed is 6.7 - 26.7 μL·min -1 , the voltage is 20 - 28 kV, the receiving distance of the roller receiver is 12 - 20 cm, and the roller rotation speed is 100 - 300 r·min -1 . The temperature of electrospinning is room temperature (20 - 30 °C), and the humidity is 30 - 50%. Preferably, the high-voltage electrospinning control parameters are as follows: the feeding speed is 20 μL·min -1 , the voltage is 24 kV, the receiving distance of the roller receiver is 18 cm, and the roller rotation speed is 200 r·min -1 ; the fibers obtained by spinning are crosslinked at a high temperature of 120 °C for 1 h.

[0008] Further, in step S3, the foaming solution is a NaBH4 solution with a concentration of 0.05 - 0.2 M (preferably 0.1 M).

[0009] Further, in step S3, the nanofibers are soaked in the foaming solution for 20 - 40 min (preferably 30 min); the time for removing air bubbles is 2 - 6 s; the freezing condition is freezing at -80 °C for 0.5 - 1.5 h; the freeze-drying time is 12 - 24 h, and the freeze-drying temperature is -70 °C to -60 °C (preferably, the time for removing air bubbles is 3 s, the freezing condition is freezing at -80 °C for 1 h; the freeze-drying time is 24 h, and the freeze-drying temperature is -67 °C).

[0010] Further, in step S4, the concentration of the muscone solution is 3.5 - 5%.

[0011] In the second aspect, the present invention provides a traditional Chinese medicine muscone sustained-release nanofiber dressing prepared according to the described method.

[0012] In the traditional Chinese medicine muscone sustained-release nanofiber dressing of the present invention, the fiber diameter is 389 ± 42 nm, the porosity is 67.7 ± 5.3%, the specific surface area is 192.6 ± 7.6 M 2 / g, and the muscone loading amount is 1.2 - 1.5 Mg / cm 2 . Under the weak alkaline (pH in the range of 7.5 - 8.0) condition of simulating diabetic wounds, the cumulative release rate in 72 h is 75.9 ± 2.1%.

[0013] Thirdly, the present invention provides the application of the traditional Chinese medicine muskone sustained-release nanofiber dressing in the preparation of products for improving blood circulation in diabetic wounds.

[0014] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) The present invention combines the active ingredient muskone of traditional Chinese medicine with nanomaterials to construct a nanofiber dressing Musc@CP-NF loaded with muskone, realizing the controllable and slow release of the muskone drug; (2) The muskone sustained-release nanofiber dressing of the present invention has the effect of inhibiting the expression of TRPV4 protein on cells, thereby inhibiting the influx of Ca 2+ 2+, reducing vasoconstriction caused by the contraction of vascular smooth muscle cells, and thus improving blood circulation; (3) The preparation method of the present invention has simple process and strong operability, and the prepared nanofiber dressing has a high porosity and a large specific surface area, which is beneficial to the loading and slow release of drugs and can meet the actual needs. Description of the Drawings

[0015] Figure 1 is the process flow chart of the present invention; Figure 2 is the SEM (A) and AFM (B) diagrams of the nanofiber carrier CP-NF obtained in Example 1 of the present invention; Figure 3 is the muskone drug loading result diagram of Musc@CP-NF obtained in Example 2 of the present invention; wherein, A: Musc loading amount; B: CP-NF; C: SEM diagram of Musc@CP-NF soaked in PBS solution for 3 days; Figure 4 is the muskone drug release behavior curve diagram of Musc@CP-NF obtained in Example 2 of the present invention; Figure 5 is the result diagram of the TRPV4 signaling pathway regulation mechanism of the present invention; wherein, A: Western Blot result of C166 cells expressing TRPV4; B: Influence of Musc@CP-NF on [Ca 2+ i 2+induced by hyperglycemia; C: Immunofluorescence staining to detect the TRPV4 expression of cells in different treatment groups; Figure 6 is the detection result diagram of intracellular Ca 2+ 2+concentration in Example 2 of the present invention; wherein, A: Immunofluorescence staining to detect Ca 2+ 2+concentration; B: Fluorescence intensity statistics of Fluo-4 AM; Figure 7 is the result of Musc@CP-NF inhibiting Ca​2+ Volcano plot and heat map of differential gene expression of inward flow Figure 8 In Example 2 of the present invention, Musc@CP-NF inhibits Ca 2+ Distribution and enrichment result map of differential genes inhibiting Ca inward flow in MF molecular function Figure 9 Result map of Musc@CP-NF obtained in Example 2 of the present invention promoting wound healing in a diabetic mouse model; wherein, A: Representative photographs of wound healing in different treatment groups at 0, 3, 6, 9, and 12 days; B: Statistical chart of healing time; C: Proportion of unhealed wounds Figure 10 Result map of Musc@CP-NF obtained in Example 2 of the present invention promoting angiogenesis in skin tissue; wherein, A: α Immunofluorescence labeling of -SMA and CD31; B: Statistical chart of blood vessel diameter Detailed implementation mode

[0016] The present invention aims to provide a traditional Chinese medicine muskone sustained-release nanofiber dressing, a preparation method thereof, and its application in improving blood circulation in diabetic wounds. The present invention combines the traditional Chinese medicine active ingredient muskone with nanomaterials to construct a chitosan hydrochloride (Chitosan)-pullulan (Pullulan) nanofiber dressing (Musc@CP-NF) loaded with traditional Chinese medicine muskone (Muscone, Musc). By inhibiting Ca 2+ inward flow, it can effectively relieve vasoconstriction, improve local blood circulation, and help provide the oxygen and nutrient supply required for wound healing

[0017] The present invention adopts the following technical solutions The present invention provides a preparation method of a traditional Chinese medicine muskone sustained-release nanofiber dressing, comprising the following steps S1. Dissolve chitosan hydrochloride and pullulan polysaccharide in acetic acid solution respectively, stir at room temperature for 6-10 h until a uniformly dispersed solution is formed, mix the two solutions in a mass ratio of chitosan hydrochloride to pullulan polysaccharide of 1:2-1:6, and continue to stir for 4-8 h to form a mixed solution with a concentration of 10-15 wt% (preferably 12.5 wt%) S2. Perform high-voltage electrospinning on the mixed solution to prepare nanofibers, and then crosslink at a high temperature of 100-135 °C for 0.5-2 h S3. Immerse the nanofibers obtained in step S2 completely in the foaming solution, wash the foamed nanofibers after stabilization, then perform degassing treatment, freeze and lyophilize to obtain porous nanofibers S4. Dissolve muscone in absolute ethanol, then soak the porous nanofibers in the muscone solution, and wait for the absolute ethanol to completely volatilize to prepare a traditional Chinese medicine muscone sustained-release nanofiber dressing.

[0018] Furthermore, in step S1, the concentration of the glacial acetic acid solution is 12 - 50% (preferably 50%).

[0019] Preferably, in step S1, the mass ratio of chitosan hydrochloride to pullulan polysaccharide is 1:4; the concentration of the chitosan hydrochloride and pullulan polysaccharide mixed solution is 12.5 wt%.

[0020] Furthermore, in step S2, the high-voltage electrospinning control parameters are as follows: the feeding speed is 6.7 - 26.7 μL·min -1 , the voltage is 20 - 28 kV, the receiving distance of the drum receiver is 12 - 20 cm, and the drum rotation speed is 100 - 300 r·min -1 ; the temperature of electrospinning is room temperature (20 - 30 °C), and the humidity is 30 - 50%.

[0021] Preferably, in step S2, the high-voltage electrospinning control parameters are as follows: the feeding speed is 20 μL·min -1 , the voltage is 24 kV, the receiving distance of the drum receiver is 18 cm, and the drum rotation speed is 200 r·min -1 ; the fibers obtained by spinning are crosslinked at a high temperature of 120 °C for 1 h.

[0022] Furthermore, in step S3, the foaming solution is a NaBH4 solution, and the concentration is 0.05 - 0.2 M, preferably the concentration is 0.1 M.

[0023] Furthermore, in step S3, the nanofibers are soaked in the foaming solution for 20 - 40 min (preferably 30 min); the time for removing air bubbles is 2 - 6 s; the freezing condition is freezing at -80 °C for 0.5 - 1.5 h; the freeze-drying time is 12 - 24 h, and the freeze-drying temperature is -60 °C to -70 °C.

[0024] Preferably, the time for removing air bubbles is 3 s, the freezing condition is freezing at -80 °C for 1 h; the freeze-drying time is 24 h, and the freeze-drying temperature is -67 °C. Furthermore, in step S4, the concentration of the ethanol solution containing muscone is 3.5 - 5%.

[0025] The present invention also provides a traditional Chinese medicine muscone sustained-release nanofiber dressing prepared according to the described method.

[0026] In the traditional Chinese medicine muscone sustained-release nanofiber dressing of the present invention, the fiber diameter is 389 ± 42 nm, the porosity is 67.7 ± 5.3%, and the muscone loading amount is 1.2 - 1.5 Mg / cm 2, under the weakly alkaline condition (pH ranging from 7.5 to 8.0) simulating diabetic wounds, the cumulative release rate at 72 h is 75.9 ± 2.1%.

[0027] The present invention also provides the application of the above-mentioned traditional Chinese medicine muskone sustained-release nanofiber dressing in the preparation of products for improving blood circulation in diabetic wounds.

[0028] The expression level of TRPV4 protein in cells treated with the traditional Chinese medicine muskone sustained-release nanofiber dressing Musc@CP-NF of the present invention is significantly reduced. Compared with the high-glucose treatment group, the expression level of TRPV4 in cells of Musc@CP-NF is reduced by 38.2 ± 1.5%; the wound healing time is shortened to 12.1 ± 0.8 days (compared with 18.7 ± 1.2 days in the control group), and the intracellular [Ca 2+ i (intracellular calcium ion concentration) decreases by 49.46 ± 3.1%.

[0029] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] The electrospinning device used in the following examples is purchased from Beijing Yongkang Leye Co., Ltd., model: ET-2535X.

[0031] The electrospinning device includes: a high-voltage power supply, a propulsion extrusion pump with controllable speed and a rotating drum receiver. The spinning solution propulsion extrusion pump is a micro-flow pump (single pump), and the flow direction of the spinning solution in the syringe to the extrusion device is controlled by the spinning solution propulsion device; the extrusion device is a single needle head; the collection device is square. Among them, the spinning solution propulsion device and the extrusion device are connected by a silica gel tube. At the same time, in order to ensure firm connection and prevent the spinning solution from overflowing due to pressure, threaded joints are respectively installed at both ends of the silica gel tube, and the inner diameter of the needle head of the extrusion device is 0.4 mm. The electrospinning device extrudes the spinning solution through the propulsion extrusion pump to form a liquid at the tip of the extrusion device. The liquid droplet changes from a spherical shape to a conical shape (i.e., "Taylor cone") under the action of an electric field, and a fiber filament is extended from the tip of the cone. It runs a relatively long distance in the electric field and finally solidifies into a fiber, which is received on the rotating drum receiver and finally crosslinked at a preset temperature.

[0032] ​Chitosan hydrochloride (Mw < 50000) and muscone were purchased from Shanghai Macklin Biochemical Co., Ltd.; pullulan polysaccharide (200 - 400 kDa) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; glacial acetic acid was purchased from Energy Chemical; sodium borohydride (NaBH4) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; phosphate buffer solution, PBS (pH 7.2 - 7.4) and CCK-8 were all purchased from Beijing Solarbio Science & Technology Co., Ltd.; high-glucose DMEM medium and fetal bovine serum were purchased from Wuhan Procell Life Science & Technology Co., Ltd.; calcium ion fluorescent probe (Fluo-4 AM) and TRPV4 antibody were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; TRPV4 agonist (GSK1016790A) and TRPV4 antagonist (HC-067047) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; mouse vascular endothelial cell C166 was purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences.

[0033] Example 1 Preparation method of a nanofiber carrier As Figure 1 shown, a preparation method of a nanofiber carrier includes the following steps: 1) Weigh a certain amount of Chitosan powder and Pullulan powder at room temperature and dissolve them in 50% glacial acetic acid solution respectively. Stir at room temperature for 8 h until a uniformly dispersed solution is formed. Mix these two solutions at a mass ratio of 1:4. The total polysaccharide concentration in the mixed solution is 12.5 wt%. Continue to stir for 6 h to finally form a stable mixed solution; 2) Use a high-voltage electrospinning instrument to prepare nanofibers. The spun fibers are cross-linked at 120 °C for 1 h. The high-voltage electrospinning instrument mainly consists of a high-voltage power supply, a propulsion extrusion pump with controllable speed, and a rotating drum receiver. First, load 9 mL of the mixed solution into a 10 mL disposable syringe, set the propulsion speed to 22.7 μL·min -1 , voltage to 24 kV, the receiving distance to 12 cm. The rotating drum receiver is used to receive the electrospinning jets, and set the rotating drum speed to 200 r·min -1 .

[0034] 3) Carefully take out the NaBH4 powder from the dryer. First add distilled water to a beaker, weigh a certain amount of NaBH4 and dissolve it in distilled water to prepare a 0.1 M NaBH4 solution. Immerse the nanofibers completely in the NaBH4 solution. After 30 min to reach stability, transfer the foamed nanofibers to a new well plate, wash them three times with distilled water, then place them in a circulating water pump vacuum pump to exhaust bubbles for 3 s, take them out and freeze them in a -80 °C refrigerator for 1 h, and then freeze-dry them in a freeze dryer for 24 h to obtain porous nanofibers CP-NF.

[0035] Due to the numerous process parameters in electrospinning, in order to efficiently and evenly arrange multi-factor and multi-level experiments, obtain comprehensive experimental results with the least number of experiments, and thus optimize the preparation process of CP-NF, three factors and three levels of the main process parameters are used, namely: the concentration of the polysaccharide mixed solution (10, 12.5, 15 wt%), the spinning voltage (20, 24, 28 kV), and the receiving distance (6, 12, 18 cm). These are combined and an orthogonal experiment is designed using the L9(3 4 ) orthogonal array to obtain 9 combinations. After electrospinning nanofibers under each combination condition, three indicators, namely fiber diameter, specific surface area, and porosity, are comprehensively evaluated, and the best combination is selected as the preparation condition for nanofibers used in subsequent experiments.

[0036] The results of the multi-factor and multi-level experiments are as follows: Table 1 Orthogonal experiment design and results

[0037] The morphology of the nanofiber carrier prepared in Example 1 was characterized by scanning electron microscopy, as shown in Figure 2 Figure A, with magnification factors of 1k, 5k, 10k, and 25k. Before morphology characterization, in order to improve the contrast of the nanofiber carrier, it was sputter-coated with gold. It can be statistically obtained from the figure that the diameter of CP-NF is about 389 nm, and the specific surface area and porosity of the drug-loaded nanomaterial in Example 1 were tested using a fully automatic physical and chemical sorption analyzer. Before the test, the drug-loaded nanomaterial was purged with nitrogen at 350 °C for 5 h to remove the vapor and impurities in it, and a surface area of 192.6 M 2 / g and a porosity of 67.7% were obtained.

[0038] The fiber height and morphology of the nanofiber carrier prepared in Example 1 were characterized by atomic force microscopy, as shown in Figure 2 Figure B. An RTESPA-3005 silicon probe (the radius of the cantilever tip is less than 10 nm) was used. Before the test, the cantilever was cleaned under an ultraviolet light source for 15 min. The tapping mode of AFM, that is, the tapping mode, was used and carried out at room temperature and in air. The line speed of AFM image acquisition was 0.3 Hz, 256 × 256 pixs, and at least two discontinuous regions of (10 - 15) μm × (10 - 15) μm were measured for each sample. The images were processed using NanoScope Analysis 1.9 software. First, forced leveling was performed, and then binomial fitting was used. The AFM images showed that the surface of the nanofibers was smooth, without obvious defects or particle attachment. The fiber height was evenly distributed, further confirming the continuity and uniformity of the fibers. Through AFM height profile analysis, the diameter distribution of the fibers was measured. The results showed that the fiber diameter was consistent with the SEM results, mainly distributed in the range of 380 - 410 nm, and the average diameter was 389 nm.

[0039] Example 2 Preparation Method of Musk-Controlled Release Nanofiber Dressing As Figure 1 shown, the preparation method of the musk-controlled release nanofiber dressing includes the following steps: Dissolve musk in absolute ethanol to obtain a musk solution with a concentration of 4.71 wt%. Subsequently, soak the CP-NF prepared in Example 1 with a size of 10.0 mm × 10.0 mm × 10.0 mm in 1 mL of the musk solution. After the absolute ethanol has completely volatilized, the Musc@CP-NF uniformly loaded with musk can be prepared.

[0040] The surface properties of Musc@CP-NF are related to the loading amount and stability of Musc. To study the drug-loading stability of Musc, fix CP-NF and Musc@CP-NF on a glass rod, place them in a beaker filled with PBS solution, and oscillate them in a shaker at a speed of 150 rpm under the condition of a 37°C water bath for 72 h. Take them out and let them dry, and observe by SEM and perform XPS analysis. As Figure 3 shown in A, drop an excessive amount of ethanol solution containing Musc onto the CP-NF nanofiber carrier. After the ethanol in the solution has completely volatilized, weigh the increased mass of CP-NF and obtain the drug-loading nanofiber dressing Musc@CP-NF with a Musc loading rate of 32.6% compared with the original mass of CP-NF. After soaking in PBS solution for 72 h, the structure of CP-NF is intact, indicating that CP-NF can stably exist in a liquid environment; some Musc particles can still be observed on the surface of Musc@CP-NF, indicating that the loaded Musc can maintain its effect for at least 3 d, ensuring the continuous exertion of the drug effect ( Figure 3 B and Figure 3 C). Use the full spectrum and fine spectrum of X-ray photoelectron spectrometer, and use Mg-K α line: 1253.6 eV, line width 0.7 eV. Among them, perform high-resolution scanning on C, O, and N elements and calculate the change in the content ratio between elements. The results are shown in Table 2. After 72 h, the C / O ratio of CP-NF is 1.86, while the C / O ratio of Musc@CP-NF, although it decreases from 3.11 to 2.37, is still significantly higher than that of CP-NF. This may be attributed to the large specific surface area and high porosity of CP-NF, which are conducive to the full adsorption of Musc. The above results indicate that Musc@CP-NF has good stability in aqueous solution, providing a basis for the drug effect of Musc at the wound site.

[0041] Table 2 XPS Element Content Analysis Table of CP-NF and Musc@CP-NF after Soaking in PBS Solution for 72 h

[0042] Musc@CP-NF was loaded into a pre-treated dialysis bag (Mw = 1000) and immersed in a container filled with 10 mL of PBS buffer solution (pH = 8.0). It was stirred under constant temperature at 37 °C. Every 24 h, 3 mL of the solution was taken out from the dialysis bag, and the absorbance (Abs) of the taken-out solution was measured with a UV-visible spectrophotometer at the maximum absorption wavelength of Musc (λmax = 280 nm). The same amount (3 mL) of fresh PBS buffer solution was replenished. At this time, the drug concentration in the PBS buffer solution was proportional to its absorbance. Therefore, by using the pre-prepared standard curve for testing, the drug concentration in the solution at time t could be obtained. Then, plotting time against drug concentration, the drug release curve of Musc@CP-NF was obtained ( Figure 4 ) showed that in the initial stage of release (0 - 24 h), Musc@CP-NF exhibited a relatively high drug release rate, and the cumulative release rate reached 33.4 ± 2.6%. This phenomenon was mainly attributed to the rapid dissociation and diffusion of the surface-adsorbed drug. In the later stage of release (24 - 72 h), the drug release rate decreased significantly, to 75.9 ± 2.1%, and tended to be stable after 72 h. This stage mainly relied on the diffusion of the drug inside Musc@CP-NF and the gradual degradation of CP-NF, indicating that it was more suitable for the treatment requirements of diabetic wounds in such a complex pathological environment.

[0043] The expression of TRPV4 in MAEC, MOVAS, C166, and C166- / - cells (gene silencing of TRPV4 by transfection with TRPV4 siRNA (siRNA sense: 5’-ggagctgaacaagaactca-3’, SEQ ID NO:1)) was detected by Western blot respectively. The results were as Figure 5 shown in A. TRPV4 was expressed in all three endothelial cells. According to the colorimetric Maker, the target band showed a double band, and the molecular weights were approximately 85 KDa and 100 Kda. Further, through the cell immunofluorescence staining technique, the effect of Musc@CP-NF on the expression level of TRPV4 protein in C166 cells was detected to reveal its mechanism of action in regulating Ca 2+ influx and improving microcirculation in diabetic wounds. A multifunctional microplate reader was used to detect the effect of Musc@CP-NF on [Ca 2+ i . The results were as Figure 5 shown in B. Under the hyperglycemic environment, the intracellular [Ca 2+ i concentration increased significantly, and Ca 2+ ​​Increased inward current. The agonist GSK1016790A (abbreviated as GSK) exacerbates Ca 2+ inward current in a concentration-dependent manner (GSK100, GSK300, and GSK500 represent the concentrations of GSK at 100, 300, and 500 nM, respectively). Conversely, the inhibitor HC067047 (abbreviated as HC) significantly inhibits Ca 2+ inward current. At the same time, Musc@CP-NF has a significant inhibitory effect on [Ca 2+ i . The results of immunofluorescence staining for detecting the expression level of TRPV4 protein showed ( Figure 5 C): Hyperglycemia can promote the expression of TRPV4. The agonist GSK300 can make the expression of TRPV4 stronger, while the inhibitor HC significantly inhibits the expression of TRPV4. At the same time, it was also found that the expression of TRPV4 in cells treated with Musc@CP-NF was significantly reduced, indicating that Musc@CP-NF has the effect of inhibiting TRPV4 on the cell membrane.

[0044] The present invention further verified the inhibitory effect of Musc@CP-NF on Ca 2+ inward current. A confocal microscope was used to observe the fluorescence intensity of Fluo-4 AM to reflect the fluorescence intensity of calcium ions. Fluo-4 AM is one of the most commonly used probes for detecting the intracellular Ca 2+ concentration. Fluo-4 AM is a fluorescent dye that can penetrate the cell membrane and belongs to the acetyl methyl ester derivative of Fluo-4. Since the fluorescence of Fluo-4 AM is extremely weak, it will not increase with the increase in Ca 2+ concentration. After entering the cell, Fluo-4 AM is cleaved by intracellular esterase to form Fluo-4. The binding of Fluo-4 and Ca 2+ can produce strong fluorescence that can be detected. As Figure 6 the results showed, the expression of TRPV4 in cells was positively correlated with the [Ca 2+ i concentration. Hyperglycemia can promote the expression of TRPV4, and correspondingly, the [Ca 2+ i concentration is higher; the addition of the inhibitor not only reduces the expression of TRPV4 but also reduces the [Ca 2+ i concentration; Musc@CP-NF can also inhibit the expression of TRPV4 and at the same time reduce the [Ca 2+ i concentration.

[0045] Analysis of the mechanism by which Musc@CP-NF inhibits Ca 2+ inward current showed that Musc@CP-NF inhibits Ca 2+ ​​​​​Effective inhibition of internal flow. Omics analysis was performed on the differential genes of C166 cells treated with Musc@CP-NF for 24 h. The overall situation of differentially expressed genes was represented by volcano plots and heatmaps ( Figure 7 ), compared with the untreated group, 376 genes were significantly up-regulated after Musc@CP-NF treatment, and the up-regulated genes had effects such as promoting cell proliferation; correspondingly, 307 genes were significantly down-regulated, and the down-regulated genes had effects such as weakening cell inflammation at the wound.

[0046] To visually display the distribution and enrichment degree of differential genes in the MF molecular function, a scatter plot was used to show the GO enrichment results ( Figure 8 ). The analysis found that the activities of voltage-gated sodium channels and calcium channels showed differences, suggesting that there might be interference in calcium ion channels.

[0047] The present invention further evaluated the application potential of muskone sustained-release nanofiber dressings in diabetic wound healing. A type II diabetic mouse model was established by combining a high-sugar and high-fat diet with multiple low-dose intraperitoneal injections of streptozotocin (STZ). STZ is a glucosamine-nitrosourea derivative, and the nitrosourea in its structure is a cytotoxin that can selectively damage β islet cells, resulting in insulin deficiency.

[0048] First, 8-week-old C57BL / 6J mice were fed a high-fat and high-sugar diet for 4 weeks to induce insulin resistance, and then an STZ solution with a dose of 40 - 50 mg·kg -1 was intraperitoneally injected (STZ was dissolved in A / B solution, where A is citric acid and B is sodium citrate dihydrate, configured with A:B = 1:1.2). After each injection, the mice were fed a normal high-fat diet and given water, fasted overnight with water supply, and then given a normal diet and water after injecting STZ the next day. After continuous injection for five days, they were continuously fed a high-fat diet for more than two weeks. Blood was taken from the tip of the tail, and the fasting blood glucose was ≥11.1 mmol·L -1 for three consecutive days, and observing that their urine was viscous and had a strong odor indicated successful modeling. This method can establish a diabetic animal model with peripheral insulin resistance and mild impairment of islet function, which is similar to the occurrence and development process of human type II diabetes, and the dosage of STZ is small, reducing the damage to other tissues.

[0049] The diabetic mice were randomly divided into four groups: a control group, a 3M group, a CP-NF group, and a Musc@CP-NF group, with 6 mice in each group. The hair on the backs of the mice was removed, and after intraperitoneal injection of avertin anesthetic, a circular full-thickness skin wound model with a diameter of 8 mm was created using a biopsy punch. The wound dressing was covered at the wound site, and a surgical waterproof adhesive patch with a hollow in the middle was covered on the wound dressing to firmly adhere the waterproof adhesive patch around the wound dressing to firmly fix it.

[0050] During the experiment, the wounds were photographed and recorded at 0, 3, 6, 9, and 12 d by digital photography. The wound healing rate was quantitatively analyzed using Image J software ( Figure 9 A). The macroscopic images of the wounds showed that the Musc@CP-NF group exhibited a significant wound-healing promoting effect. On the 3rd day after surgery, compared with the control group, the wound contraction rates of the 3M (a commercial dressing produced by 3M Company, Minnesota Mining and Manufacturing Company, USA, with a porosity of 24.5%), CP-NF, and Musc@CP-NF groups increased by 13.7%, 22.0%, and 41.2%, respectively. By the 6th day, the wound healing rate of the Musc@CP-NF group was significantly better than that of the other groups, and no obvious inflammatory reaction was observed in the wounds of this group, while different degrees of inflammatory reactions were shown in the other groups. On the 9th day, there were still 41.0 ± 3.5% and 30.8 ± 7.3% unhealed wounds in the control group and the 3M group, respectively, accompanied by persistent inflammation. It is worth noting that the wounds in the Musc@CP-NF group were completely epithelialized on the 12th day after surgery, with a complete new epidermal tissue and orderly collagen arrangement; in contrast, the wounds in the other groups were still open, accompanied by obvious tissue defects and inflammatory reactions. Statistical analysis of the wound healing rate ( Figure 9 B) showed that the Musc@CP-NF group had completely healed at the end of the experiment, while there were still 22.8 ± 4.6%, 16.4 ± 0.5%, and 6.2 ± 3.9% unhealed wounds in the control group, the 3M group, and the CP-NF group, respectively. In addition, the statistical results of the time to complete epithelialization ( Figure 9 C) further confirmed the wound-healing promoting effect of Musc@CP-NF. The average time to complete epithelialization in the Musc@CP-NF group was 11.4 ± 0.6 days, which was significantly shorter than that in the control group (15.4 ± 1.6 days), the 3M group (14.4 ± 1.4 days), and the CP-NF group (12.8 ± 1.2 days). These quantitative data were highly consistent with the aforementioned histological observation results, fully confirming the significant advantages of Musc@CP-NF in promoting diabetic wound healing.

[0051] Furthermore, immunofluorescence staining was used to evaluate angiogenesis and myofibroblast differentiation during the wound healing process ( Figure 10 A). CD31, as a specific marker for vascular endothelial cells, reflects the levels of angiogenesis and maturation. On the 9th day after surgery, new blood vessels were formed in all groups, but the vascular density in the Musc@CP-NF group was significantly higher than that in the other groups, and the vascular structure was more mature, suggesting that it may promote angiogenesis by regulating the VEGF / VEGFR2 signaling pathway, or it may improve blood circulation by regulating the Ca 2+ pathway. At the same time, α-SMA expression, and this protein is a specific marker for myofibroblast differentiation. The results are as Figure 10 shown in B, the Musc@CP-NF group α -SMA positive cell number increased significantly. These cells showed a typical contractile phenotype and formed a large number of actin stress fibers. Myofibroblasts play a key role in the wound healing process: promoting wound closure through contraction; regulating the wound microenvironment and coordinating the repair process.

[0052] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made thereto, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Preparation method of Chinese medicine muskone sustained-release nanofiber dressing, characterized in that, Including the following steps: S1. Dissolve chitosan hydrochloride and pullulan polysaccharide in glacial acetic acid solution respectively, stir at room temperature until a uniformly dispersed solution is formed to obtain chitosan hydrochloride solution and pullulan polysaccharide solution; mix the two polysaccharide solutions in proportion and continue stirring. The total polysaccharide concentration in the obtained mixed solution is 10-15 wt%, and the mass ratio of chitosan hydrochloride to pullulan polysaccharide is 1:2-1:6; S2. Perform high-voltage electrospinning on the mixed solution to prepare nanofibers, and then crosslink at a high temperature of 100-135 °C for 0.5-2 h; S3. Immerse the nanofibers obtained in step S2 completely in the foaming solution, wash the foamed nanofibers after stabilization, then perform degassing treatment, freeze and lyophilize to obtain porous nanofibers; S4. Dissolve muskone in absolute ethanol to obtain a muskone solution; then immerse the porous nanofibers in the muskone solution, and wait for the absolute ethanol to completely volatilize to obtain the traditional Chinese medicine muskone sustained-release nanofiber dressing.

2. The method according to claim 1, wherein In step S1, the concentration of the glacial acetic acid solution is 20-50%.

3. The method according to claim 1, characterized in that In step S2, the control parameters of high-voltage electrospinning are as follows: the feeding speed is 6.7 - 26.7 μL·min -1 , the voltage is 20 - 28 kV, the receiving distance of the drum receiver is 12 - 20 cm, and the drum rotation speed is 100 - 300 r·min -1 .

4. The method according to claim 1, wherein In step S3, the foaming solution is a NaBH4 solution with a concentration of 0.05-0.2 M.

5. The method according to claim 1, characterized in that, In step S3, the nanofibers are immersed in the foaming solution for 20-40 min for stabilization; the degassing time is 2-6 s; the freezing condition is freezing at -80 °C for 0.5-1.5 h; the lyophilization time is 12-24 h, and the lyophilization temperature is -70 °C to -60 °C.

6. The method according to any one of claims 1-5, characterized in that In step S4, the concentration of the muskone solution is 3.5-5%.

7. The traditional Chinese medicine muskone sustained-release nanofiber dressing prepared by the method according to any one of claims 1-6.

8. The traditional Chinese medicine muskone sustained-release nanofiber dressing according to claim 7, characterized in that, In the traditional Chinese medicine muscone sustained-release nanofiber dressing, the fiber diameter is 389 ± 42 nm, the porosity is 67.7 ± 5.3%, the specific surface area is 192.6 ± 7.6 m 2 / g, and the muscone loading amount is 1.2 - 1.5 mg / cm 2 .

9. Use of the traditional Chinese medicine muskone sustained-release nanofiber dressing according to claim 7 or 8 in the preparation of a product for improving blood circulation in diabetic wounds.

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