Application of sea cucumber-derived nano-vesicles in preparation of medicine for promoting wound repair

Abstract

The invention relates to application of sea cucumber-derived nano-vesicles in preparation of a medicine for promoting wound repair. The nano-vesicles (SC-EVs) extracted and prepared from sea cucumbers can effectively promote cell migration, scratch repair and tube forming capabilities of human skin epithelial cells (HACAT) and human skin microvascular endothelial cells (HM DEC) and inhibit cell apoptosis, so that the capability of repairing cells is achieved, and particularly the effect of repairing wound surfaces in a high glucose state or diabetes mellitus is achieved. The invention provides a new direction and thought for the application of the sea cucumber-sourced nano vesicles (SC-EVs), provides a brand new drug research and development and clinical treatment direction for wound repair, especially for solving the problem of difficult healing of wounds of patients with high glucose or diabetes, and has extremely high social value and market application prospect.

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] As a global chronic disease, the skin complications of diabetes, especially the healing of diabetic wounds, have always been an important challenge in clinical research and treatment. The wound healing process of diabetic patients is complex, involving blood sugar levels, microcirculation, neuropathy, immune function, collagen synthesis, signal pathway regulation, and angiogenesis. The continuous increase in blood sugar levels in diabetic patients provides favorable conditions for the growth of bacteria. The high-sugar environment not only promotes the growth and reproduction of bacteria, but also weakens the body's resistance to bacteria and increases the risk of infection. In addition, high blood sugar may also cause a decline in the function of immune cells, resulting in a weakening of the killing power of white blood cells in a high-sugar environment, thereby increasing the risk of infection. Microcirculation disorders often occur in diabetic patients, which is the result of the combined action of multiple factors such as vascular endothelial cell damage, hemodynamic changes, and metabolic disorders caused by long-term high blood sugar. Microcirculatory disorders can lead to insufficient blood supply, affecting the local nutrient and oxygen supply to the wound, thereby hindering the healing process of the wound.

[0003] Insufficient blood supply can lead to local hypoxia, edema and nutrient deficiency in the wound, which in turn affects the metabolic activity and proliferation ability of cells. In addition, microcirculatory disorders may also affect the transport and distribution of drugs, making it difficult for drugs to reach the wound and exert their effects. Angiogenesis is one of the key links in the wound healing process. The formation of new blood vessels can provide sufficient nutrition and oxygen supply to the wound, promote cell proliferation and migration, and thus accelerate wound healing. In the diabetic state, angiogenesis is affected by multiple factors, resulting in inhibition or abnormality of this process. Pathophysiological conditions such as hyperglycemia, hyperlipidemia and insulin resistance can interfere with the expression of vascular endothelial growth factor and the signal transduction of its receptors, thereby inhibiting the proliferation and migration ability of vascular endothelial cells. In addition, diabetes can also cause microvascular lesions, such as thickening of the microvascular basement membrane, a decrease in the number of microvessels, and the formation of microaneurysms, all of which will further hinder the angiogenesis and repair process of the wound.

[0004] As a kind of seafood with extremely high nutritional and medicinal value, sea cucumber has been widely verified in the application research of diabetes. Among them, the application of sea cucumber in diabetes management is mainly reflected in its multi-faceted benefits to diabetic patients. Sea cucumbers are rich in various nutrients, such as sea cucumber acid mucopolysaccharides, trace metal elements, sea cucumber saponins, etc. These ingredients can activate the activity of pancreatic islet cells and stabilize high blood sugar concentrations. Repair pancreatic islet function: The sea cucumber acid mucopolysaccharides in sea cucumbers have powerful repair and regeneration functions, which can slowly repair damaged and inactive pancreatic islet cells, activate and regenerate the secretion function of pancreatic islet cells, and gradually restore the pancreatic islet function to form a virtuous cycle, thereby reducing the harm of diabetes. Reduce blood viscosity: The polysaccharide components in sea cucumbers can reduce blood viscosity in the body, especially inhibit vascular aging, and help improve the blood circulation of diabetic patients. Purify blood: After entering the human body, sea cucumbers can replenish blood, purify blood, and help improve the organ function of diabetic patients. Improve immunity: The polysaccharides in sea cucumbers have immunoregulatory functions, which can improve the immunity of diabetic patients and reduce the risk of infection. 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 can 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 protein supplementation 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 wound repair in the prior art, thereby isolating and extracting sea cucumber wall-broken nanovesicles from individual sea cucumbers, and conducting functional studies on them, clarifying that they can effectively promote 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 wounds under high sugar conditions or diabetes.

[0006] In order to solve the above technical problems, the present invention is implemented 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 surface are selected from one or more of the self-healing ability of skin epithelial cells, the self-healing ability of skin microvascular endothelial cells, the migration ability of skin epithelial cells, the migration ability of skin microvascular endothelial cells, the proliferation ability of skin microvascular endothelial cells, the apoptosis level of skin microvascular endothelial cells, the tube-forming ability of skin microvascular endothelial cells, and the expression of ki67 / PCNA 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) centrifuging the sea cucumber treated solution at 1000 g for 30 min and filtering it using a 100-mesh sieve;

[0013] (3) Collect the supernatant, centrifuge at 5000 g for 10 min, and filter using a 100-mesh sieve;

[0014] (4) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter using a 0.45 μm filter;

[0015] (5) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter 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 using 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 as follows: 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 filtering in step (6).

[0021] The second aspect of the present invention provides the use of sea cucumber-derived nanovesicles in the preparation of drugs that promote 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) centrifuging the sea cucumber treated solution at 1000 g for 30 min and filtering it using a 100-mesh sieve;

[0025] (3) Collect the supernatant, centrifuge at 5000 g for 10 min, and filter using a 100-mesh sieve;

[0026] (4) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter using a 0.45 μm filter;

[0027] (5) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter 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 using 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 as follows: 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 filtering in step (6).

[0033] The 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 surface are selected from one or more of the self-healing ability of skin epithelial cells, the self-healing ability of skin microvascular endothelial cells, the migration ability of skin epithelial cells, the migration ability of skin microvascular endothelial cells, the proliferation ability of skin microvascular endothelial cells, the apoptosis level of skin microvascular endothelial cells, the tube-forming ability of skin microvascular endothelial cells, and the expression of ki67 / PCNA 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) centrifuging the sea cucumber treated solution at 1000 g for 30 min and filtering it using a 100-mesh sieve;

[0039] (3) Collect the supernatant, centrifuge at 5000 g for 10 min, and filter using a 100-mesh sieve;

[0040] (4) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter using a 0.45 μm filter;

[0041] (5) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter 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 using 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 as follows: 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 filtering 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) centrifuging the sea cucumber treated solution at 1000 g for 30 min and filtering it using a 100-mesh sieve;

[0051] (3) Collect the supernatant, centrifuge at 5000 g for 10 min, and filter using a 100-mesh sieve;

[0052] (4) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter using a 0.45 μm filter;

[0053] (5) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter 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 using 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 as follows: 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 filtering in step (6).

[0059] The 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 makes it difficult to heal skin surface injuries, especially diabetic foot disease, which has always been the focus of medical attention. As one of the complications of diabetes, diabetic foot has a long hospitalization period, high hospitalization costs, and high mortality and disability rates, which seriously affects the quality of life of patients and increases the economic burden. The wound healing process of diabetic patients is complex, involving blood sugar levels, microcirculation, neuropathy, immune function, collagen synthesis, signal 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 microvascular tumors. These lesions will further hinder the angiogenesis and repair process of the wound surface. As a type of seafood with extremely high nutritional value and medicinal value, sea cucumbers have been widely verified in the application research of diabetes, among which the application of sea cucumbers in diabetes management is mainly reflected in its multifaceted 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) scratches; in addition, the cell proliferation and cell tube formation 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 formation ability of HMDEC cells. At the same time, SC-EVs inhibit the apoptosis of HMDEC cells under high sugar conditions. The present invention also compared SC-EVs with SC-polysaccharide (sea cucumber polysaccharide) and SC-Glycosides (sea cucumber saponins) to show a positive effect on wound healing in diabetic model mice (DB DB mice). 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 development and clinical treatment for wound repair, especially for overcoming the problem of difficult wound healing in patients with high sugar or diabetes, which has extremely 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 effect 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] Fig. 9 Schematic diagram of the effect of SC-EVs on wound healing in DBDB mice.

[0071] Fig.10 Schematic diagram of the effect of SC-EVs on the expression of ki67 / PCNA in the wound tissue of DBDB mice. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used 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 figure. 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 Confirmation 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, and 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 a sea cucumber processing liquid.

[0077] (2) The sea cucumber treated liquid was centrifuged at 1000 g for 30 min and filtered using a 100-mesh sieve to remove insoluble precipitates as much 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, centrifuge at 10,000 g for 30 min, and 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, take the precipitate and resuspend it in PBS to obtain enriched SC-EVs, and finally filter it with a 0.22 μm filter to obtain a sterile SC-EVs suspension.

[0082] The SC-EVs prepared above were then taken for morphological identification, and the specific steps were 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 for 2 minutes). Use filter paper to absorb the remaining sample liquid, and there will be 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 wet copper grid float on the droplet. After 2 minutes, use filter paper to absorb the excess staining solution. After natural drying, observe under an electron microscope. SC-EVs can be seen as round or oval vesicles, like trays, with diameters ranging from 50-200nm (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 into a 1 mL syringe, connected to the machine sample port, and injected slowly and at a uniform speed 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, the effects of sea cucumber-derived nanovesicles SC-EVs on the scratch self-healing ability and cell migration ability of human skin epithelial cells (HACA T) and human skin microvascular endothelial cells (HMDEC) were studied. 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 cells were plated in 6-well plates at 10,000 cells per well and divided into experimental and control groups. 1 mg / mL 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 h. A marking pencil is used to mark the back of the culture plate. When the cell confluence reaches 90%-95%, a straight scratch is made with a gun tip along the position marked by the pencil on the back of the plate to scratch the cells.

[0089] (3) After scratching, wash each group of cells with PBS. This experiment requires at least 3 replicate wells for each group to reduce errors caused by human operation errors. After PBS washing, return the cells to the incubator for continuous culture. Use a microscope to take pictures of the same scratch position at 0h, 24h, and 48h of culture and statistically analyze the scratch differences. The results are as follows: 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 A cell suspension of 10000 to 100000 cells / mL was prepared and plated into Transwell wells, with about 10,000 to 100,000 cells per well depending on the cell size; after the cells were plated, an appropriate volume of serum-free culture medium was added (so that the culture medium in the well reaches 80 to 100 uL).

[0092] (2) SC-EVs with a final concentration of 10 ug / mL (converted according to the volume of the upper chamber liquid) 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 uL of complete culture medium was added to the bottom well plate; after 12-24 h of culture, 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 HDMEC.

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

[0094] (1) HMDEC cells were plated in a 96-well plate at 1000 cells / well and divided into 4 groups. Group 1 and Group 2 used 500 mg / mL 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 in normal DEME complete medium and served as the control group; 5 replicate wells were set 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 ug / mL); an equal volume of fresh culture medium was added to Group 2 and Group 4 as the control group.

[0096] (3) Add 10uL CCK8 detection solution to each well, incubate at 37℃ in the dark for 2h, and measure the absorbance at 450nm on an ELISA reader. The test results are as follows: Figure 6 The results showed that the proliferation of HMDEC cells could be inhibited under high glucose conditions, while SC-EVs could significantly promote the proliferation of HMDEC cells. Moreover, 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 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 / mL of cell suspension.

[0099] (2) The cell suspension was plated into a 6-well plate at a rate of 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, where 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 using normal DEME complete medium and served as the control group; 3 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 ug / 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 hours, 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 minutes. 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 FITC-Annexin V and 5μL PI in turn, vortex gently to mix, and react at room temperature in the dark for 15 minutes. Add 400μL 1*Binding Buffer to each tube, transfer to flow tubes, mark according to group, and perform flow analysis on the machine. 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, which was significantly lower than that of the control group under high glucose conditions (**p < 0.01, VS group 2 (HG group)).

[0103] Furthermore, the effect of SC-EVs on HMDEC cell tube formation under high glucose was studied. The specific steps are as follows:

[0104] (1) HMDEC cells with good growth status 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 / mL of cell suspension.

[0105] (2) The cell suspension was plated into a 6-well plate at a rate of 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, where 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 using normal DEME complete medium and served as the control group; 3 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 ug / 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 inoculated into a 96-well plate that had been pre-coated with high-concentration growth factor matrix gel. The cells could be inoculated only after the matrix gel had solidified. After inoculating the cells, the volume was supplemented to 100 uL. After 6 hours, photos were taken and statistics were obtained. The test results were as follows: Figure 8 The results showed that high glucose could inhibit the tube-forming ability of HMDEC cells, while SC-EVs could significantly promote the tube-forming ability of HMDEC cells; and under high glucose conditions, the tube-forming ability of cells treated with SC-EVs could be 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 respectively. PBS was dripped on the wounds of mice in Group 1 as the control group (NC group), 10 mg / mL sea cucumber saponins (SC-Glycosides) were dripped on the wounds of mice in Group 2, 10 mg / mL sea cucumber polysaccharides (SC-polysaccharide) were dripped on the wounds of mice in Group 3, and 10 mg / mL sea cucumber-derived nanovesicles (SC-EVs) were dripped on 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 isolated 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, and sea cucumber saponins had a certain effect on wound healing; sea cucumber polysaccharides and sea cucumber-derived nanovesicles could effectively promote wound repair, and sea cucumber-derived nanovesicles had a more significant effect on wound healing, which was significantly better than the wound healing promoting effect of sea cucumber polysaccharides and sea cucumber saponins; the expression of ki67 / PCNA in immunohistochemistry also indicated 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 therapeutic 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 healing wounds in patients with high sugar or diabetes, and have extremely high social value and market application prospects.

[0115] The above specific implementation method part specifically introduces the analytical method involved in the present invention. It should be noted that the above introduction is only to help those skilled in the art better understand the method and ideas of the present invention, rather than limiting the relevant content. Without departing from the principle of the present invention, those skilled in the art may also make appropriate adjustments or modifications to the present invention, and the above adjustments and modifications shall also fall within the scope of protection of the present invention.

Claims

1. Application of sea cucumber-derived nanovesicles in the preparation of drugs for promoting wound repair and / or improving physiological indicators related to wounds.

2. The use according to claim 1, characterized in that: The wound surface is a wound surface caused by diabetes and / or high sugar state.

3. The use according to claim 1, characterized in that: The physiological indicators related to the wound surface are selected from one or more of the following: the self-healing ability of skin epithelial cells, the self-healing ability of skin microvascular endothelial cells, the migration ability of skin epithelial cells, the migration ability of skin microvascular endothelial cells, the proliferation ability of skin microvascular endothelial cells, the apoptosis level of skin microvascular endothelial cells, the tube-forming ability of skin microvascular endothelial cells, and the expression of ki67 / PCNA in skin tissue.

4. 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.

5. The pharmaceutical composition according to claim 4, characterized in that The wound surface is a wound surface caused by diabetes and / or high sugar state.

6. The pharmaceutical composition according to claim 4, characterized in that The physiological indicators related to the wound surface are selected from one or more of the following: the self-healing ability of skin epithelial cells, the self-healing ability of skin microvascular endothelial cells, the migration ability of skin epithelial cells, the migration ability of skin microvascular endothelial cells, the proliferation ability of skin microvascular endothelial cells, the apoptosis level of skin microvascular endothelial cells, the tube-forming ability of skin microvascular endothelial cells, and the expression of ki67 / PCNA in skin tissue.

7. The pharmaceutical composition according to claim 4, 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.

8. Application of sea cucumber-derived nanovesicles in the preparation of drugs that promote angiogenesis.

9. A method for preparing sea cucumber-derived nanovesicles, characterized in that: The steps include: (1) washing and soaking the sea cucumber and performing a wall-breaking process to obtain a sea cucumber processing solution; (2) centrifuging the sea cucumber treated solution at 1000 g for 30 min and filtering it using a 100-mesh sieve; (3) Collect the supernatant, centrifuge at 5000 g for 10 min, and filter using a 100-mesh sieve; (4) Collect the supernatant, centrifuge at 10,000 g for 30 min, and filter using a 0.45 μm filter; (5) Collect the supernatant, centrifuge at 10,000 g for 30 min, and 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 using a 0.22 μm filter.

10. The sea cucumber-derived nanovesicles prepared according to the preparation method of claim 9.

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