Application of sea cucumber-derived nanovesicles in the preparation of drugs for promoting wound repair

Abstract

The present invention relates to the application of sea cucumber-derived nanovesicles in the preparation of drugs for promoting wound repair. The nanovesicles (SC-EVs) extracted and prepared from sea cucumbers of the present invention can effectively promote the cell migration, scratch repair, and tube formation ability of human skin epithelial cells (HACAT) and human skin microvascular endothelial cells (HM DEC), and inhibit cell apoptosis, thereby achieving the ability to repair cells, especially the repair effect on wounds under high sugar conditions or diabetic wounds. The present invention provides a new direction and idea for the application of sea cucumber-derived nanovesicles (SC-EVs), and provides a new direction for drug research and development and clinical treatment for wound repair, especially for overcoming the problem of difficult wound healing in patients with high sugar or diabetes, and has extremely high social value and market application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of sea cucumber-derived nanovesicles in wound repair under diabetic / high sugar conditions. Background Art

[0002] Diabetes, a global chronic disease, presents significant challenges in clinical research and treatment for its skin complications, particularly diabetic wound healing. The wound healing process in diabetic patients is complex, involving multiple factors, including blood glucose levels, microcirculation, neuropathy, immune function, collagen synthesis, signaling pathway regulation, and angiogenesis. The persistently elevated blood glucose levels in diabetic patients create favorable conditions for bacterial growth. This high-glucose environment not only promotes bacterial growth and reproduction but also weakens the body's defenses against bacteria, increasing the risk of infection. Furthermore, hyperglycemia can lead to decreased immune cell function, reducing the killing capacity of white blood cells in high-glucose environments and exacerbating the risk of infection. Microcirculatory impairment is common in diabetic patients, resulting from the combined effects of multiple factors, including endothelial cell damage, hemodynamic changes, and metabolic disturbances caused by chronic hyperglycemia. This impaired microcirculation leads to insufficient blood supply, impairing local nutrient and oxygen delivery to the wound, and thus hindering wound healing.

[0003] Inadequate blood supply to the wound surface can lead to localized hypoxia, edema, and nutrient deficiency, which in turn impairs cellular metabolic activity and proliferation. Furthermore, impaired microcirculation can impair the transport and distribution of drugs, making it difficult for them to reach the wound and be effective. Angiogenesis is a key component of wound healing. The formation of new blood vessels provides an adequate supply of nutrients and oxygen to the wound surface, promoting cell proliferation and migration, thereby accelerating wound healing. In diabetic patients, angiogenesis is influenced by multiple factors, leading to inhibition or abnormalities in this process. Pathological conditions such as hyperglycemia, hyperlipidemia, and insulin resistance can interfere with the expression of vascular endothelial growth factor and its receptor signaling, thereby inhibiting the proliferation and migration of endothelial cells. Furthermore, diabetes can cause microvascular lesions, such as thickening of the microvascular basement membrane, a decrease in microvessel count, and the formation of microaneurysms. These lesions further impede wound angiogenesis and repair.

[0004] As a seafood with exceptional nutritional and medicinal value, sea cucumbers have been extensively validated in diabetes research. Their use in diabetes management is primarily reflected in their multifaceted benefits for diabetic patients. Sea cucumbers are rich in various nutrients, such as acidic mucopolysaccharides, trace metal elements, and sea cucumber saponins. These ingredients can activate pancreatic islet cell activity and stabilize high blood sugar levels. Repairing pancreatic function: The acidic mucopolysaccharides in sea cucumbers possess powerful repair and regenerative properties. They can slowly repair damaged and inactive pancreatic islet cells, activating and regenerating their secretory function, gradually restoring pancreatic function and creating a virtuous cycle, thereby alleviating the harmful effects of diabetes. Reducing blood viscosity: The polysaccharides in sea cucumbers can reduce blood viscosity, particularly by inhibiting vascular aging, helping to improve blood circulation in diabetic patients. Purifying the blood: Sea cucumbers replenish and purify the blood, helping to improve organ function in diabetic patients. Boosting immunity: The polysaccharides in sea cucumbers have immunomodulatory properties, boosting the immune system and reducing the risk of infection in diabetic patients. Prevent complications: The selenium in sea cucumbers helps improve pancreatic function and prevent the occurrence of diabetes-related complications, such as cardiovascular disease. In addition, the unsaturated fatty acids in sea cucumbers also help lower blood lipids, improve blood circulation, and further prevent diabetes from complicating cardiovascular diseases. Rich in nutrients: Sea cucumbers are rich in nutrients such as protein, multiple amino acids, vitamins, and minerals, which can provide comprehensive and balanced nutritional support for diabetic patients and enhance their physical fitness. Supplement essential amino acids: Sea cucumbers contain essential amino acids for the human body, which are very beneficial for supplementing protein and help diabetic patients maintain a good nutritional status. However, there are few reports in the existing technology on the role of sea cucumber-derived nanovesicles in wound repair. Summary of the Invention

[0005] The purpose of the present invention is to address the problems existing in the wound repair technology in the prior art, and thus isolate and extract sea cucumber wall-broken nanovesicles from individual sea cucumbers, and conduct functional studies on them. It is clear that they can effectively promote the cell migration, scratch repair, and tube formation ability of human skin epithelial cells (HACAT) and human skin microvascular endothelial cells (HMDEC), and inhibit cell apoptosis, thereby achieving the ability to repair cells, especially the repair effect on wounds under high sugar conditions or diabetes.

[0006] In order to solve the above technical problems, the present invention is achieved through the following technical solutions.

[0007] A first aspect of the present invention provides the use of extracellular vesicles derived from sea cucumber (SC-EVs) in the preparation of a drug for promoting wound repair and / or improving physiological indicators related to the wound.

[0008] Preferably, the wound surface is a wound surface caused by diabetes and / or high sugar state.

[0009] Preferably, the physiological indicators related to the wound are selected from one or more of the following: skin epithelial cell self-healing ability, skin microvascular endothelial cell self-healing ability, skin epithelial cell migration ability, skin microvascular endothelial cell migration ability, skin microvascular endothelial cell proliferation ability, skin microvascular endothelial cell apoptosis level, skin microvascular endothelial cell tube-forming ability, and ki67 / PCNA expression in skin tissue.

[0010] Preferably, the sea cucumber-derived nanovesicles are prepared by the following method:

[0011] (1) washing and soaking the sea cucumber and performing a wall-breaking process to obtain a sea cucumber processing solution;

[0012] (2) The sea cucumber treated solution was centrifuged at 1000 g for 30 min and filtered using a 100-mesh sieve;

[0013] (3) The supernatant was collected and centrifuged at 5000 g for 10 min, and filtered through a 100-mesh sieve;

[0014] (4) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.45 μm filter;

[0015] (5) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.22 μm filter;

[0016] (6) Collect the supernatant and centrifuge at 150,000 g for 90 min. Discard the supernatant, resuspend the precipitate, and filter it through a 0.22 μm filter.

[0017] Preferably, the washing in step (1) is specifically: washing with tap water for 1-10 times, and then washing with ultrapure water for 1-5 times.

[0018] Preferably, the soaking in step (1) is specifically: soaking in ultrapure water for 10-120 minutes.

[0019] Preferably, PBS is used for resuspending in step (6).

[0020] Preferably, a 0.22 μm filter is used for filtration in step (6).

[0021] A second aspect of the present invention provides the use of sea cucumber-derived nanovesicles in the preparation of a drug that promotes angiogenesis.

[0022] Preferably, the sea cucumber-derived nanovesicles are prepared by the following method:

[0023] (1) washing and soaking the sea cucumber and performing a wall-breaking process to obtain a sea cucumber processing solution;

[0024] (2) The sea cucumber treated solution was centrifuged at 1000 g for 30 min and filtered using a 100-mesh sieve;

[0025] (3) The supernatant was collected and centrifuged at 5000 g for 10 min, and filtered through a 100-mesh sieve;

[0026] (4) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.45 μm filter;

[0027] (5) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.22 μm filter;

[0028] (6) Collect the supernatant and centrifuge at 150,000 g for 90 min. Discard the supernatant, resuspend the precipitate, and filter it through a 0.22 μm filter.

[0029] Preferably, the washing in step (1) is specifically: washing with tap water for 1-10 times, and then washing with ultrapure water for 1-5 times.

[0030] Preferably, the soaking in step (1) is specifically: soaking in ultrapure water for 10-120 minutes.

[0031] Preferably, PBS is used for resuspending in step (6).

[0032] Preferably, a 0.22 μm filter is used for filtration in step (6).

[0033] A third aspect of the present invention provides a pharmaceutical composition for promoting wound repair and / or improving physiological indicators related to the wound, comprising sea cucumber-derived nanovesicles and a pharmaceutically acceptable carrier.

[0034] Preferably, the wound surface is a wound surface caused by diabetes and / or high sugar state.

[0035] Preferably, the physiological indicators related to the wound are selected from one or more of the following: skin epithelial cell self-healing ability, skin microvascular endothelial cell self-healing ability, skin epithelial cell migration ability, skin microvascular endothelial cell migration ability, skin microvascular endothelial cell proliferation ability, skin microvascular endothelial cell apoptosis level, skin microvascular endothelial cell tube-forming ability, and ki67 / PCNA expression in skin tissue.

[0036] Preferably, the sea cucumber-derived nanovesicles are prepared by the following method:

[0037] (1) washing and soaking the sea cucumber and performing a wall-breaking process to obtain a sea cucumber processing solution;

[0038] (2) The sea cucumber treated solution was centrifuged at 1000 g for 30 min and filtered using a 100-mesh sieve;

[0039] (3) The supernatant was collected and centrifuged at 5000 g for 10 min, and filtered through a 100-mesh sieve;

[0040] (4) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.45 μm filter;

[0041] (5) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.22 μm filter;

[0042] (6) Collect the supernatant and centrifuge at 150,000 g for 90 min. Discard the supernatant, resuspend the precipitate, and filter it through a 0.22 μm filter.

[0043] Preferably, the washing in step (1) is specifically: washing with tap water for 1-10 times, and then washing with ultrapure water for 1-5 times.

[0044] Preferably, the soaking in step (1) is specifically: soaking in ultrapure water for 10-120 minutes.

[0045] Preferably, PBS is used for resuspending in step (6).

[0046] Preferably, a 0.22 μm filter is used for filtration in step (6).

[0047] Preferably, the pharmaceutically acceptable carrier is selected from one or more of a filler, a binder, a lubricant, a disintegrant, a flavoring agent, a colorant, an antioxidant, an antibacterial agent, a chelating agent, a surfactant, and a solvent.

[0048] A fourth aspect of the present invention provides a method for preparing sea cucumber-derived nanovesicles, comprising the following steps:

[0049] (1) washing and soaking the sea cucumber and performing a wall-breaking process to obtain a sea cucumber processing solution;

[0050] (2) The sea cucumber treated solution was centrifuged at 1000 g for 30 min and filtered using a 100-mesh sieve;

[0051] (3) The supernatant was collected and centrifuged at 5000 g for 10 min, and filtered through a 100-mesh sieve;

[0052] (4) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.45 μm filter;

[0053] (5) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.22 μm filter;

[0054] (6) Collect the supernatant and centrifuge at 150,000 g for 90 min. Discard the supernatant, resuspend the precipitate, and filter it through a 0.22 μm filter.

[0055] Preferably, the washing in step (1) is specifically: washing with tap water for 1-10 times, and then washing with ultrapure water for 1-5 times.

[0056] Preferably, the soaking in step (1) is specifically: soaking in ultrapure water for 10-120 minutes.

[0057] Preferably, PBS is used for resuspending in step (6).

[0058] Preferably, a 0.22 μm filter is used for filtration in step (6).

[0059] A fifth aspect of the present invention provides sea cucumber-derived nanovesicles prepared according to the above preparation method.

[0060] Due to its pathophysiological characteristics, diabetes mellitus makes wound healing difficult after skin damage, particularly diabetic foot disease, a topic of ongoing medical concern. As a complication of diabetes, diabetic foot disease is associated with prolonged hospitalizations, high costs, and high mortality and disability rates, severely impacting patients' quality of life and increasing the economic burden. The wound healing process in diabetic patients is complex, involving multiple factors, including blood glucose levels, microcirculation, neuropathy, immune function, collagen synthesis, signaling pathway regulation, and angiogenesis.

[0061] Diabetes or high sugar levels usually cause microvascular lesions, such as thickening of the microvascular basement membrane, a decrease in the number of microvessels, and the formation of microaneurysms. These lesions will further hinder the angiogenesis and repair process of the wound. As a type of seafood with extremely high nutritional and medicinal value, sea cucumbers have been widely verified in the application research of diabetes. Among them, the application of sea cucumbers in diabetes management is mainly reflected in its multi-faceted benefits to diabetic patients. In order to promote angiogenesis and repair of wounds in diabetic patients, the present invention separates and extracts nanovesicles after the sea cucumber wall is broken from individual sea cucumbers, thereby providing a treatment plan that effectively improves the wound repair of diabetic patients. The sea cucumber-derived nanovesicles SC-EVs extracted by the present invention can promote the self-healing ability of human skin epithelial cells (HACAT) and human skin microvascular endothelial cells (HMDEC); in addition, the cell proliferation and cell tube-forming ability of HMDEC cells can be inhibited under high sugar conditions, and SC-EVs can restore this inhibitory effect and promote the proliferation and tube-forming ability of HMDEC cells. At the same time, SC-EVs inhibit the apoptosis of HMD EC cells under high sugar conditions. The present study also demonstrated a positive effect of SC-EVs on wound healing in diabetic mice (DB DB mice) by comparing SC-polysaccharide (sea cucumber polysaccharide) and SC-Glycosides (sea cucumber saponins). This study provides a new direction and approach for the application of sea cucumber-derived nanovesicles (SC-EVs), offering a new direction for drug development and clinical treatment for wound repair, especially for overcoming the difficulty in wound healing in patients with high blood sugar or diabetes. This approach has high social value and market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the extraction steps of sea cucumber-derived nanovesicles.

[0063] Figure 2 Schematic diagram of electron microscopy detection results of sea cucumber-derived nanovesicles.

[0064] Figure 3 Schematic diagram of the particle size analysis results of SC-EVs using nanoparticle tracking analysis technology.

[0065] Figure 4 Schematic diagram of the effects of SC-EVs on the scratch self-healing ability of HACAT cells and HDMEC.

[0066] Figure 5 Schematic diagram of the effect of SC-EVs on the migration ability of HACAT cells and HDMEC.

[0067] Figure 6 Schematic diagram of the effect of SC-EVs on HMDEC cell proliferation.

[0068] Figure 7 Schematic diagram of the effect of SC-EVs on HMDEC cell apoptosis.

[0069] Figure 8 Schematic diagram of the effect of SC-EVs on the tube-forming ability of HMDEC cells.

[0070] Figure 9 Schematic diagram of the effects of SC-EVs on wound healing in DBDB mice.

[0071] Figure 10 Schematic diagram of the effects of SC-EVs on ki67 / PCNA expression in wound tissues of DBDB mice. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0073] Unless otherwise specified, the cell lines listed in the context of the present invention, including human skin epithelial cells (HACAT) and human skin microvascular endothelial cells (HMDEC), were purchased from Shanghai Fuheng Biological Company and cultured according to conventional methods in the art. The reagents used in the present invention were all commercially available, and the DBDB mice used were purchased from Youda (Guangzhou) Biotechnology Co., Ltd. The experimental methods used in the present invention, such as vesicle extraction, cell culture, cell migration experiments, cell clone formation experiments, cell proliferation experiments, etc., are all conventional methods and techniques in the art. Representative results selected from repeated biological experiments are presented in the context figures, and the data are displayed as mean±SD and mean±SEM as specified in the figures. All experiments were repeated at least three times. The data were analyzed using GraphPad Prism 8.0 or SP SS22.0 software. Conventional medical statistical methods such as t-test, Wilcoxon test, Pearson coefficient, chi-square test, analysis of variance, etc. were used to compare the mean differences between two or more groups. p<0.05 is considered a significant difference.

[0074] Example 1 confirmed with sea cucumber-derived nanovesicles SC-EVs

[0075] First, the preparation and confirmation of sea cucumber-derived nanovesicles SC-EVs are shown in the following flow chart: Figure 1 As shown, the specific steps include:

[0076] (1) Remove the internal organs and head and tail of freshly purchased sea cucumbers, retain the middle trunk, wash them repeatedly with tap water for 5 times, then wash them with ultrapure water for 3 times, add ultrapure water twice the volume of the sea cucumber and soak them for 1 hour, then use a wall breaking machine to break the wall to obtain sea cucumber processing liquid.

[0077] (2) The sea cucumber treated liquid was centrifuged at 1000 g for 30 min and filtered through a 100-mesh sieve to remove as much insoluble precipitate as possible.

[0078] (3) The supernatant was collected and centrifuged at 5000 g for 10 min, and filtered through a 100-mesh sieve to further remove the precipitate.

[0079] (4) The supernatant was collected and centrifuged at 10,000 g for 30 min, and filtered using a 0.45 μm filter to remove insoluble small particles.

[0080] (5) Collect the supernatant and centrifuge at 10,000 g for 30 min, then filter using a 0.22 μm filter.

[0081] (6) Collect the supernatant and centrifuge at 150,000 g for 90 min (Hitachi ultracentrifuge). Discard the supernatant and resuspend the precipitate in PBS to obtain enriched SC-EVs. Finally, filter with a 0.22 μm filter to obtain a sterile SC-EV suspension.

[0082] The SC-EVs prepared above were then subjected to morphological identification. The specific steps are as follows:

[0083] (1) Negative staining: Dilute SC-EVs with PBS at a ratio of 1:100, take 20uL after dilution, drop it on a clean glass slide, and place it on a grid (float on the droplet to pick up the sample, time 2 minutes). Filter paper absorbs the remaining sample liquid, and there is only a thin layer of sample liquid film on the grid. Drop the diluted SC-EVs negative staining solution on the glass slide, and let the undried copper grid float on the droplet. After 2 minutes, use filter paper to absorb the excess dye. After natural drying, observe under an electron microscope. SC-EVs can be seen as round or oval vesicles, like trays, with diameters ranging from 50 to 200nm (see Figure 2 ).

[0084] (2) Nanoparticle Tracking Analysis (NTA) technology was used to measure and quantify the particle size of SC-EVs: SC-EVs were diluted to 1 mL in an appropriate proportion, and the DDNV dilution was drawn up with a 1 mL syringe. The syringe was connected to the sample port of the machine and injected slowly and uniformly to measure the particle size and number of SC-EVs. The results showed that the particle size of SC-EVs was mainly distributed between 50 and 200 nm, which was consistent with the above results, and fewer impurity peaks were found (see Figure 3), which proves that the membrane microparticles extracted by the preparation method of the present invention have good effect.

[0085] Example 2 In vitro activity study of SC-EVs

[0086] First, we studied the effects of sea cucumber-derived nanovesicles (SC-EVs) on the scratch healing and cell migration abilities of human skin epithelial cells (HACA T) and human skin microvascular endothelial cells (HMDEC). The specific steps of the scratch experiment are as follows:

[0087] (1) Select HACAT cells and HMDEC cells with good growth status to prepare a concentration of 1*10 5 The cell suspension was plated at 10,000 cells per well in a 6-well plate and divided into an experimental group and a control group. 1 mg / mL of SC-EVs was added to the experimental group, and an equal volume of PBS was added to the control group.

[0088] (2) After the cells are laid and the drug is added for stimulation, the plates are returned to the incubator and cultured for 48 hours. A marking pencil is used to mark the back of the culture plate. When the cell confluence reaches 90%-95%, the tip of the gun is used to make straight scratches along the position marked by the pencil on the back of the plate to scratch the cells.

[0089] (3) After scratching, each group of cells was washed with PBS. At least three replicates were required for each group to reduce errors caused by human operation errors. After washing with PBS, the cells were returned to the incubator for continuous culture. Microscopes were used to take pictures of the same scratch position at 0 h, 24 h, and 48 h of culture, and the scratch differences were statistically analyzed. The results are shown in Figure 2. Figure 4 The results showed that SC-EVs can effectively promote the scratch self-healing ability of HACAT cells and HDMEC.

[0090] The specific steps of the cell migration experiment are as follows:

[0091] (1) Select HACAT cells and HMDEC cells with good growth status to prepare a concentration of 1*10 5 Prepare a cell suspension of 10000 to 100,000 cells / mL and plate them into Transwell wells, with approximately 10,000 to 100,000 cells per well depending on cell size. After plating the cells, add an appropriate volume of serum-free culture medium (so that the culture medium in the well reaches 80-100 uL).

[0092] (2) SC-EVs with a final concentration of 10 μg / mL (converted to the liquid volume of the upper chamber) were added to the upper chamber of the experimental group, and an equal volume of PBS was added to the upper chamber of the control group; 400-450 μL of complete culture medium was added to the bottom well plate; after culturing for 12-24 h, the cells were fixed, stained, and counted. The test results were as follows: Figure 5The results showed that SC-EVs could effectively promote the migration ability of HACAT cells and HDMECs.

[0093] The effects of SC-EVs on HMDEC cell proliferation and apoptosis under high glucose were then studied. The cell proliferation experimental steps were as follows:

[0094] (1) HMDEC cells were plated into 96-well plates at 1000 cells / well and divided into 4 groups. Groups 1 and 2 were cultured with 500 mg / mL glucose as the stimulation concentration for the high glucose cell model and served as the diabetes model group; Groups 3 and 4 were cultured with normal DEME complete medium and served as the control group; 5 replicate wells were set up in each group.

[0095] (2) After 12 h of culture, the culture medium of each group was removed, and fresh culture medium containing SC-EVs was added to Group 1 and Group 3 (the concentration of SC-EVs in each well after addition was 10 μg / mL); an equal volume of fresh culture medium was added to Group 2 and Group 4 as the control group.

[0096] (3) Add 10 μL of CCK8 detection solution to each well, incubate at 37°C in the dark for 2 h, and measure the absorbance at 450 nm on a microplate reader. The test results are as follows: Figure 6 The results showed that high glucose could inhibit the proliferation of HMDEC cells; while SC-EVs could significantly promote the proliferation of HMDEC cells; and under high glucose conditions, the cells treated with SC-EVs could recover to a level close to that of normal cells, which was significantly higher than that of the control group under high glucose conditions (**p < 0.01, VS group 2 (HG group)).

[0097] The specific steps of the cell apoptosis experiment are as follows:

[0098] (1) HMDEC cells in good growth condition were digested with trypsin and digested with complete medium. The cells were centrifuged at 1200 rpm for 5 min and the supernatant was removed. An appropriate amount of culture medium was added to resuspend the cell pellet. An appropriate amount of cell suspension was taken and counted using a cell counter to prepare 2*10 5 cells / mL of cell suspension.

[0099] (2) The cell suspension was plated into a 6-well plate at 2 mL / well, and the plate was placed in an incubator for normal culture. After 12 h, the 6-well plate was taken out and divided into 4 groups. Group 1 and Group 2 used 500 mg / mL of glucose stimulation concentration as the stimulation concentration of the high-glucose cell model and served as the diabetes model group. Group 3 and Group 4 were cultured with normal DEME complete medium and served as the control group. Three replicate wells were set up in each group.

[0100] (3) After 12 h of culture, the culture medium of each group was removed, and fresh culture medium containing SC-EVs was added to Group 1 and Group 3 (the concentration of SC-EVs in each well after addition was 10 μg / mL); an equal volume of fresh culture medium was added to Group 2 and Group 4 as the control group.

[0101] (4) After culturing for 24 h, the HMDEC cells in the well plate were digested with EDTA-free trypsin, and the cells were collected by centrifugation at 1000 rpm for 5 min. The cells were transferred to EP tubes, labeled according to the groups, and washed twice with pre-cooled PBS.

[0102] (5) Add 1*Binding Buffer to resuspend the cells so that the cell concentration is not less than 1.0*10 6 Pipette 100μL of the above cell suspension into another EP tube, add 5μL of FITC-Annexin V and 5μL of PI in sequence, vortex gently to mix, and react at room temperature in the dark for 15 minutes. Add 400μL of 1*Binding Buffer to each tube, transfer to flow cytometry tubes, mark according to group, and perform flow cytometry analysis. The test results are as follows Figure 7 The results showed that high glucose can promote the apoptosis of HMDEC cells, while SC-EVs can significantly inhibit the apoptosis of HMDEC cells; and under high glucose conditions, the apoptosis level of cells treated with SC-EVs was significantly reduced, significantly lower than that of the control group under high glucose conditions (**p < 0.01, VS group 2 (HG group)).

[0103] Furthermore, we investigated the effect of SC-EVs on HMDEC cell tube formation under high glucose conditions. The specific steps were as follows:

[0104] (1) HMDEC cells in good growth condition were digested with trypsin and digested with complete medium. The supernatant was removed by centrifugation at 1200 rpm for 5 min. An appropriate amount of culture medium was added to resuspend the cell pellet. An appropriate amount of cell suspension was taken and counted using a cell counter to prepare 2*10 5 cells / mL of cell suspension.

[0105] (2) The cell suspension was plated into a 6-well plate at 2 mL / well, and the plate was placed in an incubator for normal culture. After 12 h, the 6-well plate was taken out and divided into 4 groups. Group 1 and Group 2 used 500 mg / mL of glucose stimulation concentration as the stimulation concentration of the high-glucose cell model and served as the diabetes model group. Group 3 and Group 4 were cultured with normal DEME complete medium and served as the control group. Three replicate wells were set up in each group.

[0106] (3) After 12 h of culture, the culture medium of each group was removed, and fresh culture medium containing SC-EVs was added to Group 1 and Group 3 (the concentration of SC-EVs in each well after addition was 10 μg / mL); an equal volume of fresh culture medium was added to Group 2 and Group 4 as the control group.

[0107] (4) After 24 hours of continuous culture, the HMDEC cells in the plate were digested with EDTA-free trypsin, and the cells were collected by centrifugation at 1000 rpm for 5 minutes. The cells were transferred to EP tubes, labeled according to the groups, and washed twice with pre-cooled PBS. 10,000 cells per well were seeded into a 96-well plate that had been pre-coated with a high-concentration growth factor matrix gel. It was necessary to make sure that the matrix gel had solidified before seeding the cells. After seeding the cells, the volume was replenished to 100 uL. After 6 hours, photos were taken and statistics were taken. The test results were as follows: Figure 8 The results showed that high glucose inhibited the tube-forming ability of HMDEC cells, while SC-EVs significantly promoted the tube-forming ability of HMDEC cells. Moreover, under high glucose conditions, the tube-forming ability of cells treated with SC-EVs was restored to a level close to that of normal cells, which was significantly higher than that of the control group under high glucose conditions (**p < 0.01, VS group 2 (HG group)).

[0108] Example 3 In vivo activity study of SC-EVs

[0109] The diabetic mouse model (DBDB mouse) (purchased from Nanjing Model Animal Center) was used to study the promoting effect of sea cucumber-derived nanovesicles SC-Evs on wound healing. The specific steps are as follows:

[0110] (1) Using a skin puncture device, deep skin samples of the same size were taken from the skin surface of DBDB mice to form fixed wounds. The mice were then randomly divided into 4 groups, with 6 mice in each group.

[0111] (2) The wounds of mice in each group were treated separately. PBS was dripped onto the wounds of mice in group 1 as the control group (NC group), 10 mg / mL sea cucumber saponins (SC-Glycosides) were dripped onto the wounds of mice in group 2, 10 mg / mL sea cucumber polysaccharides (SC-polysaccharide) were dripped onto the wounds of mice in group 3, and 10 mg / mL sea cucumber-derived nanovesicles (SC-EVs) were dripped onto the wounds of mice in group 4. The treatment was repeated 3 times a day for 1 week, and the healing of the wounds was observed.

[0112] (3) After 2 weeks, the mice were killed, the wound skin tissue was separated by dissection, and the expression of Ki67 / PCNA in the skin tissue was analyzed by immunohistochemical staining to understand the skin healing status.

[0113] Test results such as Figure 9-10The results showed that the wounds of DBDB mice were difficult to heal, but sea cucumber saponins had a certain effect on wound healing. Sea cucumber polysaccharides and sea cucumber-derived nanovesicles could effectively promote wound repair, and the promotion effect of sea cucumber-derived nanovesicles on wound healing was more significant, significantly better than that of sea cucumber polysaccharides and sea cucumber saponins. The expression of Ki67 / PCNA in immunohistochemistry also suggested that SC-EVs treatment could effectively promote the rapid proliferation of tissue cells.

[0114] From the above, it can be clearly seen that the present invention has found through a large number of studies that sea cucumber-derived nanovesicles are a substance that can effectively promote wound repair under diabetic / high sugar conditions; by removing the internal organs and head and tail of fresh sea cucumbers, retaining the middle trunk and washing, filtering and extracting sea cucumber-derived nanovesicles SC-EVs, it can effectively promote the cell migration, scratch repair, and tube formation of human skin epithelial cells (HACAT) and human skin microvascular endothelial cells (HMDEC), and inhibit cell apoptosis, thereby achieving the ability to repair cells, especially the repair effect on high sugar conditions or diabetic wounds. The present invention also compared SC-polysaccharide (sea cucumber polysaccharide) and SC-Glycosides (sea cucumber saponins) through SC-EVs, and clarified that SC-EVs have a very significant positive effect on wound healing. In view of the complexity and multifactorial nature of the difficulty in healing diabetic wounds, the sea cucumber-derived nanovesicles SC-Evs extracted in the present invention provide a new direction and idea for wound repair and treatment applications under diabetic / high sugar conditions, and provide a new direction for drug development and clinical treatment for wound repair, especially for overcoming the difficulty in wound healing in patients with high sugar or diabetes. It has extremely high social value and market application prospects.

[0115] The above detailed description of the analytical methods involved in the present invention provides a detailed introduction. It should be noted that the above description is intended solely to help those skilled in the art better understand the methods and concepts of the present invention, and is not intended to limit the relevant content. Without departing from the principles of the present invention, those skilled in the art may make appropriate adjustments or modifications to the present invention, and such adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. The use of sea cucumber-derived nanovesicles in the preparation of a drug for promoting wound repair, characterized in that: The sea cucumber-derived nanovesicles are prepared by the following method: (1) washing and soaking the sea cucumber and then breaking the sea cucumber wall to obtain a sea cucumber processing liquid; (2) Centrifuge the sea cucumber treated solution at 1000 g for 30 min and filter through a 100-mesh sieve; (3) Collect the supernatant and centrifuge at 5000g for 10 min, then filter through a 100-mesh sieve; (4) Collect the supernatant and centrifuge at 10,000 g for 30 min, then filter using a 0.45 μm filter; (5) Collect the supernatant and centrifuge at 10,000 g for 30 min, then filter using a 0.22 μm filter; (6) Collect the supernatant and centrifuge at 150,000 g for 90 min. Discard the supernatant, resuspend the precipitate, and filter it through a 0.22 μm filter.

2. The use according to claim 1, characterized in that The wound surface is a wound surface under high sugar conditions.

3. A pharmaceutical composition for promoting wound repair and / or improving physiological indicators related to wounds, characterized in that: The invention comprises sea cucumber-derived nanovesicles and a pharmaceutically acceptable carrier; The sea cucumber-derived nanovesicles are prepared by the following method: (1) washing and soaking the sea cucumber and then breaking the sea cucumber wall to obtain a sea cucumber processing liquid; (2) Centrifuge the sea cucumber treated solution at 1000 g for 30 min and filter through a 100-mesh sieve; (3) Collect the supernatant and centrifuge at 5000g for 10 min, then filter through a 100-mesh sieve; (4) Collect the supernatant and centrifuge at 10,000 g for 30 min, then filter using a 0.45 μm filter; (5) Collect the supernatant and centrifuge at 10,000 g for 30 min, then filter using a 0.22 μm filter; (6) Collect the supernatant and centrifuge at 150,000 g for 90 min. Discard the supernatant, resuspend the precipitate, and filter it through a 0.22 μm filter.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutically acceptable carrier is selected from one or more of a filler, a binder, a lubricant, a disintegrant, a flavoring agent, a colorant, an antioxidant, an antibacterial agent, a chelating agent, a surfactant, and a solvent.

5. A method for preparing sea cucumber-derived nanovesicles, characterized in that: The steps include: (1) washing and soaking the sea cucumber and then breaking the sea cucumber wall to obtain a sea cucumber processing liquid; (2) Centrifuge the sea cucumber treated solution at 1000 g for 30 min and filter through a 100-mesh sieve; (3) Collect the supernatant and centrifuge at 5000g for 10 min, then filter through a 100-mesh sieve; (4) Collect the supernatant and centrifuge at 10,000 g for 30 min, then filter using a 0.45 μm filter; (5) Collect the supernatant and centrifuge at 10,000 g for 30 min, then filter using a 0.22 μm filter; (6) Collect the supernatant and centrifuge at 150,000 g for 90 min. Discard the supernatant, resuspend the precipitate, and filter it through a 0.22 μm filter.

6. The sea cucumber-derived nanovesicles prepared by the preparation method according to claim 5.

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