Exosome from clematis filamentosa dunn, preparation method and application

Abstract

The invention belongs to the technical field of nano biology, and relates to an exosome from clematis filamentosa dunn, a preparation method and application. The invention specifically relates to an exosome derived from clematis filamentosa dunn, a preparation method thereof and an application of the exosome in preparation of drugs for treating chronic inflammation and promoting healing of diabetic wounds. According to the preparation method of the clematis filamentosa dunn exosome, the high-purity and high-activity exosome is obtained by optimizing process parameters and combining technologies such as overspeed gradient centrifugation and ultrafiltration, and the defects in specific extraction and activity maintenance in the prior art are overcome. The Clematis filamentosa Dunn exosome provided by the invention has the functions of inflammation regulation, angiogenesis and tissue repair, and has good effects of relieving inflammation and curing chronic wounds of diabetes mellitus. Therefore, the method has good application prospect and important social value.

Description

Technical Field

[0001] The present invention belongs to the field of nanobiotechnology, and relates to the research and development of plant-derived exosomes in related applications. Specifically, it relates to a method for preparing exosomes derived from Akebia trifoliata (Thunb.) Koidz. var. leiocarpa (Hand.-Mazz.) W. T. Wang & P. K. Hsiao and their application in the preparation of drugs for treating chronic inflammation and promoting the healing of diabetic wounds. Background Art

[0002] Plant exosomes are nanoscale vesicles secreted by plant cells, with a diameter of about 30 - 150 nm, having a lipid bilayer membrane structure and being rich in various bioactive components such as proteins, nucleic acids, lipids, and polysaccharides. These exosomes play an important role in intercellular material transport, information transmission, and regulation of physiological processes. In recent years, it has been found that plant exosomes can not only function in plants but also have an impact on other organisms through cross-kingdom transmission, laying a foundation for their application in medicine and biology. With the in-depth study of plant exosomes, the application of exosomes from different plant sources in drug delivery and disease treatment has gradually become a hot topic. For example, ginseng exosomes, due to their rich content of ginsenosides, exhibit activities such as anti-inflammation and anti-aging; grapefruit exosomes are rich in naringin and have significant antioxidant and anti-tumor effects. Plant exosomes, due to their natural source, low immunogenicity, high biocompatibility, etc., have been considered as ideal candidates for novel drug delivery carriers and have good application prospects.

[0003] Chronic diabetic wounds (such as diabetic foot ulcers) are one of the common and difficult-to-treat complications of diabetes. The main reason for their formation is that diabetic patients are in a hyperglycemic state for a long time, leading to vascular lesions, nerve damage, and immune dysfunction. Their characteristics include slow healing, easy infection, and high recurrence rate, seriously affecting the quality of life of patients and may lead to serious consequences such as limb amputation. Wound healing is a complex process, including an inflammatory phase, a proliferative phase, and a remodeling phase. In chronic diabetic wounds, due to metabolic disorders and low immune function, the body's inflammatory response is often in a state of overactivation, resulting in the wound entering a persistent chronic inflammatory state, which hinders the normal tissue repair process. Therefore, controlling the inflammatory response and regulating the wound microenvironment are the keys to promoting wound healing. The current treatment of chronic diabetic wounds mainly includes blood glucose control, wound debridement, anti-infection treatment, and local wound-healing measures. However, these methods have limitations in treatment effects, such as insufficient efficacy and significant side effects. Therefore, developing new drugs targeting the inflammatory response and wound microenvironment has important value. Summary of the Invention

[0004] To solve the current technical problem of the lack of drugs for treating chronic inflammation, especially the lack of drugs for treating chronic diabetic wounds; by exploring the treasure house of traditional Chinese medicine, the present invention discovers that the extract of Clematis filamentosa Dunn in traditional Chinese medicine has various biological activities such as anti-inflammatory, antioxidant, immunomodulatory and anti-tumor effects; further in-depth research finds that Clematis filamentosa exosomes have better anti-inflammatory effects and can promote wound healing. Therefore, the following specific technical solutions are proposed to solve the above technical problems. (Clematis filamentosa-derived exosomes are hereinafter referred to as CDNVs).

[0005] The present invention discloses a method for extracting and purifying Clematis filamentosa exosomes efficiently, aiming to obtain exosomes with high purity and high activity, and overcome the deficiencies in specific extraction and activity maintenance in the prior art. By optimizing process parameters and combining technologies such as ultrafiltration and ultracentrifugation in a density gradient, high-quality preparation of exosomes is ensured. In addition, the present invention verifies the functional characteristics of Clematis filamentosa exosomes in inflammation regulation, angiogenesis and tissue repair, and explores their application potential in the treatment of chronic diabetic wounds, providing an innovative treatment strategy for this field, with important academic value and industrialization prospects.

[0006] To achieve the above object, the present invention provides a method for preparing Clematis filamentosa exosomes, and the specific steps include:

[0007] 1) Exosome extraction: Mix the washed and dried Clematis filamentosa leaves with PBS, and slowly stir at low temperature for not less than 10 hours; use a mechanical crushing device to crush the above mixture; filter the crushed mixture through a filter screen, collect the juice and put it into a centrifuge tube and place it in an ice bath;

[0008] 2) Exosome purification: Centrifuge the collected juice at a centrifugal speed of 3000 - 5000g for 10 - 30 minutes to remove large particle impurities; take the supernatant, and then centrifuge it at a speed of 130000 - 150000g for 1 - 3 hours with an ultra-high-speed centrifuge to separate exosome particles from the supernatant; resuspend the obtained exosome precipitate in PBS, and then sequentially add sucrose solutions with concentrations of 8%, 15%, 30%, 45%, 60% (g / v) for density gradient centrifugation, and centrifuge at an ultra-high speed of 130000 - 150000g for 0.5 - 2 hours; extract exosomes from the interface layer between the 30% and 45% sucrose layers, wash them 2 - 3 times with PBS, and then use an ultrafiltration tube with a molecular weight cut-off of 30 kDa to centrifuge at a centrifugal speed of 3000 - 5000g for 0.5 - 1 hour to obtain the prepared exosomes.

[0009] Preferably, the leaves of Akebia trifoliata var. australis are pretreated as follows: the harvested leaves of Akebia trifoliata var. australis are washed with sterile water or deionized water for no less than 3 times, and the washed leaves are placed in a ventilated environment to dry naturally. During the drying process, the temperature is controlled at 20-30°C, and the relative humidity is maintained at 50%-70%.

[0010] Further, during the exosome extraction process in step 1), the dried leaves of Akebia trifoliata var. australis are mixed with PBS with a weight 2-5 times that of the leaves and a pH value of 6.0-7.5, and slowly stirred at 4-10°C for 12-16 hours; the mixture is broken in a cycle of working for 1 minute and resting for 1 minute for 5 times; the broken mixture is filtered through a 30-mesh filter, and the juice is collected and put into a centrifuge tube and placed in an ice bath.

[0011] The present invention also provides exosomes derived from Akebia trifoliata var. australis obtained by the above preparation method.

[0012] The surface potential of the exosomes derived from Akebia trifoliata var. australis obtained by the above preparation method is -7.21±1.6 mV, the average particle size is about 86.57±2.3 nm, and the PDI value is 0.2; the results of lipidomics analysis show that the content of diacylglycerol in the exosomes is about 16%, the content of monogalactosyldiacylglycerol is about 10%, while the contents of phospholipids such as phosphatidylinositol, phosphatidylcholine, digalactosyldiacylglycerol and phosphatidylethanolamine are basically the same, all about 8%.

[0013] The present invention also provides the application of the exosomes derived from Akebia trifoliata var. australis in the preparation of drugs for treating and alleviating chronic inflammation and promoting diabetic wound healing.

[0014] The drug prepared from the exosomes derived from Akebia trifoliata var. australis described above is a preparation prepared with the exosomes derived from Akebia trifoliata var. australis as the main active ingredient and plus pharmaceutically common excipients or auxiliary ingredients.

[0015] Further, the preparation includes liquid preparations, solid preparations or semi-solid preparations.

[0016] Furthermore, the liquid preparation includes any one of oral liquid preparations and injection preparations; the solid preparation includes any one of tablets, granule preparations, powder preparations and capsule preparations; the semi-solid preparation includes any one of ointments, creams, gels and suppositories.

[0017] Beneficial effects: The preparation method of the exosomes of Akebia trifoliata var. australis provided by the present invention obtains exosomes with high purity and high activity by optimizing process parameters and combining technologies such as ultracentrifugation and ultrafiltration, overcoming the deficiencies of the prior art in specific extraction and activity maintenance. The exosomes of Akebia trifoliata var. australis provided by the present invention have the functions of inflammation regulation, angiogenesis and tissue repair, and have good effects on alleviating inflammation and curing chronic diabetic wounds. Description of the Drawings

[0018] Figure 1 It is a characterization diagram of exosomes derived from

[0019] Figure 2 It is an analysis diagram of the protein and lipid components of exosomes derived from

[0020] Figure 3 It is an evaluation diagram of the cell compatibility of exosomes derived from

[0021] Figure 4 It is an intracellular distribution diagram of exosomes derived from

[0022] Figure 5 It is an evaluation diagram of the in vitro inhibition of macrophage inflammatory response by exosomes derived from

[0023] Figure 6 It is an evaluation diagram of the promotion of diabetic chronic wound healing by exosomes derived from

[0024] Figure 7 It is an effect diagram of the inhibition of diabetic chronic wound inflammation by exosomes derived from Detailed Description of the Invention

[0025] The present invention will be further described in detail below in conjunction with the embodiments and the drawings. However, the embodiments of the present invention are not limited to the following content.

[0026] The following embodiments 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 embodiments are conventional means well known to those skilled in the art, and the reagents used can be commercially purchased.

[0027] Example 1 Preparation Method of Exosomes Derived from

[0028] This example provides a method for preparing exosomes from the leaves of

[0029] Pretreatment of the leaves of : Take 5 g of the leaves of , and wash them repeatedly with sterile water or deionized water to thoroughly remove the dust and microorganisms attached to their surfaces. After washing, the leaves of are naturally dried to ensure that there is no residual moisture on the leaf surface.

[0030] Initial extraction: The dried leaves of Gymnema sylvestre were thoroughly mixed with 10 mL of PBS (pH = 7.0) and incubated for 12 hours to facilitate the release of active ingredients. Subsequently, the mixture was placed in a juicer for crushing. The operation process was to stop for 1 minute after crushing for 1 minute, and this operation was repeated 5 times to ensure complete extraction. The crushed mixture was filtered through a 30-mesh sieve to remove coarse residues. The filtrate was collected and dispensed into centrifuge tubes and placed on ice for later use.

[0031] Centrifugation: The collected filtrate of Gymnema sylvestre was subjected to the following centrifugation steps in sequence:

[0032] Centrifuge at 3000g for 30 minutes and collect the supernatant;

[0033] Centrifuge the supernatant again at 10000g for 1.5 hours and collect the new supernatant;

[0034] Use an ultra-high-speed centrifuge to centrifuge the obtained supernatant at 150000g for 2.5 hours. After centrifugation, collect the precipitate part to obtain the preliminarily extracted exosome precipitate.

[0035] Sucrose gradient purification: To further purify exosomes, the sucrose gradient centrifugation method was adopted. The specific operation was as follows: Sucrose solutions were prepared at 8%, 30%, 45%, and 60% (g / v) respectively and layered into centrifuge tubes in sequence. Subsequently, ultra-high-speed centrifuge at 150000g for 1.5 hours. Collect the liquid between the 30% and 45% sucrose layers and aspirate 4 mL of purified Gymnema sylvestre-derived exosomes (CDNVs).

[0036] Removing sucrose: Add 5 volumes of PBS to the purified exosome liquid and wash it repeatedly through an ultrafiltration tube to remove sucrose. Finally, replace the solution with PBS. The obtained suspension was further filtered through a 0.22 μm microporous membrane and then through a 100 nm polycarbonate membrane to finally obtain high-purity Gymnema sylvestre-derived exosomes.

[0037] Exosome characterization: Characterize the extracted CDNVs in terms of particle size and surface potential and other properties:

[0038] The yield of CDNVs measured by the BCA method was 0.89 mg / g;

[0039] The detection results of the particle size analyzer showed that the surface potential of CDNVs was -7.21 ± 1.6 mV, the average particle size was about 86.57 ± 2.3 nm, and the PDI value was 0.2 (see Figure 1 A);

[0040] The observation results of transmission electron microscopy showed that the extracted vesicles were round and their sizes were consistent with the detection results of the particle size analyzer (see Figure 1 B).

[0041] Through the above steps, highly pure exosomes derived from *Akebia trifoliata* (Thunb.) Koidz. can be prepared, and their physicochemical properties can be effectively characterized.

[0042] Example 2 Proteomics and Lipidomics Analysis of Exosomes Derived from *Akebia trifoliata* (Thunb.) Koidz.

[0043] Lipid samples (2 mg) derived from CDNVs were sent to Beijing Bio-Tech Pack Technology Co., Ltd. (Beijing, China) for lipidomics analysis. Specifically, a triple quadrupole mass spectrometer (Q-TRAP, Applied Biosystems, California, USA) was used to study the lipid composition of CDNVs. Lipidomics data were expressed as a percentage of the total signal of molecular species after normalization to internal standards of the same lipid class. For proteomics analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate CDNVs proteins. The obtained bands were transported to Beijing Bio-Tech Pack Technology Co., Ltd. by dry ice. Subsequently, an Orbitrap mass spectrometer (Orbitrap, Thermo Fisher Scientific, Bremen, Germany) was used to qualitatively and quantitatively analyze proteins by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0044] The protein composition profile of CDNVs was identified by LC-MS / MS, and proteins were annotated and classified based on the Gene Ontology (GO) functional database. The results showed that a total of 1,892 genes were identified, and these genes were divided into different functional categories: among them, 291 genes were involved in 10 different molecular functions, mainly concentrated in catalytic activity and molecular binding functions; 920 genes were related to the cellular components of organelle anatomical entities and protein complexes; in addition, 513 genes were identified as participating in biochemical processes, which were further refined into 10 molecular events, mainly concentrated in cellular processes and metabolic processes ( Figure 2 A).

[0045] Through KEGG pathway analysis, it was found that 18 proteins in CDNVs were related to signal pathways associated with metabolism ( Figure 2 B). Among them, the pyruvate metabolism and arginine metabolism pathways play important roles in antioxidant and anti-inflammatory processes. Reductive molecules such as NADH produced by pyruvate metabolism can participate in the cellular antioxidant defense system. Peroxisomes, as important detoxifying structures within cells, can reduce oxidative damage by decomposing harmful substances such as hydrogen peroxide. These results provide important insights into the potential mechanisms of exosomes derived from *Akebia trifoliata* (Thunb.) Koidz. (CDNVs) in inhibiting oxidative stress and anti-inflammatory responses.

[0046] Lipidomics analysis results showed that the content of diacylglycerol (DG) in CDNVs exosomes accounted for about 16%, monogalactosyldiacylglycerol (MGDG) accounted for about 10%, while the contents of phospholipids such as phosphatidylinositol (PI), phosphatidylcholine (PC), digalactosyldiacylglycerol (DGDG) and phosphatidylethanolamine (PE) were basically equivalent, all about 8%. Among them, PI, PC and PE are important phospholipids involved in the antioxidant and anti-inflammatory processes, and mainly play roles by regulating cell signaling pathways and maintaining membrane structure and function ( Figure 2 C).

[0047] Example 3 Cytocompatibility evaluation of exosomes derived from Clematis armandii Franch

[0048] RAW264.7 cells were seeded in 96-well plates at a density of 10 4 cells / well and cultured overnight in an incubator containing 5% CO 2 . The exosome suspension derived from Clematis armandii Franch obtained in Example 1 was diluted with DMEM medium to prepare sample dilutions with different concentrations (2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL). The cultured RAW264.7 cells were treated with sample dilutions at different concentrations for 24 hours or 48 hours. Subsequently, 10 μL of CCK-8 reagent was added to each well and further incubated in an incubator at 37°C and 5% CO 2 for 2 hours. The absorbance value (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. Cell viability (%) = [(A 1 -A 0 ) / A 0 ×100%. Where: A 0 : absorbance value of the blank control, A 1 : absorbance value of the sample treatment group.

[0049] The results of the cytotoxicity test showed that when the exosome concentration did not exceed 200 μg / mL, after treatment with CDNVs for 24 hours or 48 hours, the viability of RAW264.7 cells remained above 80%( Figure 3 ). This result indicates that exosomes derived from Clematis armandii Franch (CDNVs) have good cytocompatibility.

[0050] Example 4 Intracellular distribution of exosomes derived from Clematis armandii Franch

[0051] RAW264.7 cells were seeded in confocal dishes at a density of 10 5 cells / well and placed in 5% CO 2Cultured overnight in an incubator. Subsequently, RAW264.7 cells in culture were treated with exosomes derived from Aristolochia fangchi (prepared in Example 1, concentration 20 μg / mL) labeled with Fluorescein-DHPE for 24 hours. After aspirating the culture medium, the cells were washed three times with PBS buffer, with each washing time being 5 minutes, and the operation needed to be carried out in the dark. 4% paraformaldehyde was added to fix the cells for 15 minutes; subsequently, the cells were washed three times with PBS again. 10 μM of lysosome stain was added and incubated at room temperature for 20 minutes; subsequently, the cells were washed three times with PBS. An anti-fluorescence quencher was added, and observations and photographs were taken through a confocal microscope (LSM 880 with AiryScan, Carl Zeiss) in a short time. In microscope detection, the excitation and emission wavelength parameters were set: green light channel Ex488, Em535; red light channel Ex543, Em611. The analysis of the obtained images was completed using ZEN 2.3 (Blue Edition, Carl Zeiss) software.

[0052] Through experimental observations, it was found that CDNVs could be effectively absorbed by RAW264.7 cells. After treatment with CDNVs for 4 hours or 8 hours, a large number of DHPE-labeled CDNVs could be detected inside the cells ( Figure 4 ). Further observations showed that most CDNVs did not co-localize with lysosomes, indicating that CDNVs endocytosed into cells could escape lysosomal degradation. Quantitative analysis of fluorescence intensity showed that the fluorescence signal of CDNVs gradually increased with the prolongation of the incubation time ( Figure 4 , scale bar = 50 μm).

[0053] Example 5 Evaluation of the in vitro anti-inflammatory and antioxidant properties of exosomes derived from Aristolochia fangchi

[0054] Detection of intracellular ROS. The oxidation-sensitive fluorescent probe DCFH-DA was used to detect the level of reactive oxygen species (ROS) in RAW264.7 cells. The specific steps were as follows: RAW264.7 cells were stimulated with LPS (100 ng / mL) to induce the production of reactive oxygen species. The stimulated cells were treated with a dilution of exosomes derived from Aristolochia fangchi (20 μg / mL) for 24 hours. After the treatment, the cells were washed three times with PBS. According to the instructions of the kit manufacturer, the cells were incubated with DCFH-DA (10 μmol / L) at 37 °C for 20 minutes. 10,000 gated cell events were detected using a flow cytometer (ACEA NovoCyte TM , USA), and the fluorescence intensity of DCFH-DA was recorded to evaluate the level of intracellular ROS.

[0055] Detection of inflammatory factor genes: RAW264.7 cells were seeded in a 6-well plate at a density of 10 5cells and placed in an incubator with 5% CO 2 overnight. After changing the medium, LPS (100 ng / mL) was added and the cells were cultured for another 12 hours to induce the formation of M1 macrophages. The cells were treated with CDNVs dilution (concentration of 20 μg / mL) for 24 hours. Total cellular RNA was extracted, and the mRNA expression levels of inflammatory factors TNF-α, IL-6, IL-1β, and iNOS were detected by real-time quantitative PCR (qPCR).

[0056] Since oxidative damage mediated by reactive oxygen species (ROS) and overexpression of inflammatory factors can accelerate the occurrence of chronic wounds, in this study, RAW264.7 cells were treated with LPS to establish an M1 macrophage model with overexpression of ROS and inflammatory factors. Further treatment with CDNVs was performed to evaluate its antioxidant and anti-inflammatory effects.

[0057] The results showed that in RAW264.7 cells stimulated with LPS, the ROS level increased significantly. After treatment with CDNVs, flow cytometry detection found that the fluorescence signal intensity of intracellular ROS decreased significantly, indicating that CDNVs had a good effect on inhibiting ROS production. Under LPS stimulation, the mRNA levels of inflammatory factors TNF-α, IL-6, IL-1β, and iNOS in RAW264.7 cells increased significantly. After treatment with CDNVs, qPCR detection showed that the mRNA levels of these inflammatory factors decreased significantly ( Figure 5 ). In summary, exosomes derived from Clematis armandii Franch. (CDNVs) can significantly inhibit the overexpression of ROS and the over-release of inflammatory factors in macrophages stimulated by LPS. This indicates that CDNVs have a good effect in alleviating oxidative damage of macrophages and inhibiting excessive inflammatory responses, further verifying its potential application value in improving the treatment of refractory wounds.

[0058] Example 6 Effect of Exosomes Derived from Clematis armandii Franch. on Promoting Diabetic Wound Healing

[0059] Establish a diabetic wound model. Diabetic model: Male Balb / c mice (8 weeks old), intraperitoneally injected with 100 μl of streptozotocin (STZ) (50 mg / kg, Sigma-Aldrich Corp., St. Louis, MO, USA). After 5 days, the fasting blood glucose level was measured in venous blood using a blood glucose meter (Sinocare, China) (after 12 hours of fasting). When the blood glucose level continuously exceeded 14 mmol / L (two fasting blood glucose measurements), the mice were evaluated and considered to have diabetes. Two weeks after the start of STZ injection, these diabetic mice were used to establish a wound model. Establish a wound model: As described previously, an excisional wound model was established in STZ-induced diabetic mice (10 weeks old, male Balb / c). Briefly, the mice were anesthetized with sodium pentobarbital (10 mg / kg or 100 μL, 2 - 3%), then the hair on the back was shaved, washed with povidone-iodine solution and cleaned with alcohol swabs. A circular dorsal wound with a diameter of 7 mm (full-thickness skin) (area ~ 38.465 mm 2 ) was made on the back of each animal using a punch (surgical scissors). In the model group, 150 μL of normal saline was applied, and in the treatment group, a sample drug solution of 200 μg / mL was applied to the back. The drug was administered at 3 pm every day, once a day for 14 consecutive days. The healing degree of the back wound was photographed every other day. At the end of the treatment, the damaged skin was collected, fixed with paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 μm. Hematoxylin and eosin (H&E) staining was performed using a NanoZoomer S360 (Hamamatsu Photonics K.K., Japan) to evaluate the overall tissue morphology.

[0060] The results showed that, compared with the blank group (treated with an equal volume of PBS), the CDNVs treatment group significantly accelerated wound healing. On the 14th day, the wound area of the mice in the treatment group was significantly smaller than that in the blank group, showing a good healing effect ( Figure 6 ). The tissue sections stained with HE on the 14th day showed that the epidermis in the CDNVs treatment group was almost completely repaired, while there were still significant pathological defects in the blank control group; the results of Masson's trichrome staining showed that the level of collagen deposition in the CDNVs group was significantly higher than that in the control group. As the main component of the extracellular matrix (ECM), collagen plays a key role in tissue regeneration and ECM remodeling. The increase in collagen deposition indicates that CDNVs promoted the process of re-epithelialization. In summary, CDNVs effectively promoted the repair of diabetic wounds by accelerating collagen deposition and epithelialization, showing important potential for clinical application.

[0061] Example 7: Exosomes derived from Clematis glycinoides Maxim. promote the anti-inflammatory and repair effects of diabetic wounds

[0062] The expression levels of inflammatory factor TNF-α, repair-promoting factor TGF-β, and angiogenesis-promoting factor VEGF were detected by real-time fluorescence quantitative PCR (qPCR). Meanwhile, the expressions of M1 and M2 macrophage markers iNOS and Arg-1 were measured.

[0063] Compared with the blank group (treated with an equal volume of PBS), the CDNVs treatment group significantly reduced the expressions of TNF-α and iNOS, indicating that CDNVs play a significant role in inhibiting local wound inflammation and reducing the number of M1 macrophages. In addition, the CDNVs treatment group significantly upregulated the expressions of TGF-β, VEGF, and Arg-1, further indicating that CDNVs can enhance wound healing by promoting M2 macrophage polarization ( Figure 7 A).

[0064] Immunohistochemistry (IHC) analysis further evaluated the expressions of specific protein markers, including TNF-α, CD86, CD206, and CD31. By quantitatively analyzing the proportion of positive staining using ImageJ software, the results showed that the CDNVs treatment group significantly reduced the expression of TNF-α at the wound site compared with the blank group, inhibited the expression of the M1 macrophage marker CD86, promoted the expression of the M2 macrophage marker CD206, and increased the expression of the vascular endothelial marker CD31 ( Figure 7 B).

[0065] These results indicate that CDNVs play an anti-inflammatory and repair-promoting role in diabetic wounds by regulating the inflammatory response and promoting angiogenesis, and have potential clinical application prospects.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing exosomes derived from Atractylodes macrocephala, characterized in that: The following steps are involved: 1) Exosome extraction: Wash and dry the leaves of Atractylodes macrocephala and mix them with PBS, and stir slowly at low temperature for at least 10 hours; The mixture is crushed by mechanical crushing equipment; the crushed mixture is filtered through a filter, the collected juice is put into a centrifuge tube and placed in an ice bath; 2) exosome purification: the collected juice is centrifuged at a centrifugal speed of 3000-5000g for 10-30 minutes to remove large particle impurities; The supernatant was taken and then centrifuged at 130000-150000g for 1-3 hours using an ultrahigh speed centrifuge to separate exosome particles from the supernatant; the obtained exosome precipitate was resuspended in PBS, and then sucrose solutions of 8%, 15%, 30%, 45%, and 60% (g / v) were added in sequence for density gradient centrifugation, and ultrahigh speed centrifugation was performed at 130000-150000g for 0.5-2 hours; the exosomes were extracted from the interface layer between the 30% and 45% sucrose layers, washed 2-3 times with PBS, and then centrifuged at 3000-5000g for 0.5-1 hour using an ultrafiltration tube with a molecular weight cutoff of 30kDa to obtain the prepared exosomes.

2. The preparation method according to claim 1, characterized in that: Pre-treat the Atractylodes lancea leaves: Use sterile water or deionized water to wash the harvested Atractylodes lancea leaves for no less than 3 times, place the washed leaves in a ventilated environment to dry naturally, and during the drying process, control the temperature at 20-30°C and the relative humidity at 50%-70%.

3. The preparation method according to claim 2, characterized in that: In step 1) of the exosome extraction process, the dried Acanthocephala leaves are mixed with 2-5 times their weight of PBS with a pH value of 6.0-7.5, and slowly stirred at a temperature of 4-10° C. for 12-16 hours; the mixture is crushed by a cycle of 1 minute working and 1 minute resting for 5 times; the crushed mixture is filtered through a 30-mesh filter, and the juice is collected and put into a centrifuge tube and placed in an ice bath.

4. Exosomes derived from Atractylodes macrocephala prepared by the method according to any one of claims 1 to 3.

5. Use of the exosomes derived from Atractylodes macrocephala as claimed in claim 4 in the preparation of drugs for treating chronic inflammation.

6. Use of the exosomes derived from Atractylodes macrocephala as claimed in claim 4 in the preparation of a drug for promoting diabetic wound healing.

7. The use of exosomes derived from Atractylodes macrocephala in the preparation of medicines according to claims 5-6, characterized in that: The medicine is a preparation prepared by taking exosomes derived from Atractylodes macrocephala as the main active ingredient and adding auxiliary materials or auxiliary ingredients commonly used in pharmacy.

8. The use of exosomes derived from Atractylodes macrocephala in the preparation of medicines according to claim 7, characterized in that: The preparations include liquid preparations, solid preparations or semisolid preparations.

9. The use of exosomes derived from Atractylodes macrocephala in the preparation of medicines according to claim 8, characterized in that: The liquid preparation includes any one of an oral liquid preparation and an injection preparation.

10. The use of exosomes derived from Atractylodes macrocephala in the preparation of medicines according to claim 8, characterized in that: The solid preparation includes any one of tablets, granule preparations, powder preparations, and capsule preparations; the semi-solid preparation includes any one of ointments, creams, gels, and suppositories.

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