Application of lemon exosomes in preparing medicine for preventing, alleviating or treating skin ulcers

Abstract

The present invention relates to the use of lemon exosomes in the preparation of a drug for preventing, alleviating, or treating skin ulcers. The invention creatively extracts and purifies exosomes from lemons and studies their physiological effects. Lemon exosomes can reduce the expression of inflammatory factors in macrophages, promote the healing of skin ulcers, lower blood sugar, and promote skin angiogenesis. Lemon exosomes have the advantages of good efficacy, simple raw material acquisition, and minimal toxic and side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the use of lemon exosomes in preparing a medicine for preventing, alleviating or treating skin ulcers. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by abnormally high blood sugar levels, called hyperglycemia, which is characterized by many complications and a high mortality rate. Chronic non-healing wounds are one of the main complications of diabetes, accounting for 19% to 34% of diabetic patients. Traditional Chinese medicine classifies diabetic wounds into the categories of "diabetes", "sores", and "ulcers". The causes are divided into internal and external factors. The internal factors are mainly "emotional injuries, improper diet, sexual injuries", etc., and the external factors are mainly "six evil toxins, special poisons", etc. The pathogenesis is mainly attributed to "stagnation of qi and blood, obstruction of meridians, blood stasis and damage to the body, and damage caused by corruption". At present, conventional treatments for diabetic wounds include blood sugar control, plant transplantation, surgical debridement, and the addition of anti-inflammatory drugs and growth factors to wound dressings, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).

[0003] Plant-derived extracellular vesicles (EVs) have emerged as an emerging frontier in therapeutics and targeted drug delivery. They offer unique advantages, such as safety, the potential for large-scale production, and intrinsic therapeutic activity against specific diseases. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide the use of lemon exosomes in the preparation of a medicament for preventing, alleviating or treating skin ulcers.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides use of lemon exosomes in the preparation of a medicament for preventing, alleviating or treating skin ulcers.

[0007] This study creatively extracts and purifies lemon exosomes and investigates their physiological effects, demonstrating their ability to reduce the expression of inflammatory factors in macrophages, promote the healing of skin ulcers, lower blood sugar, and promote skin angiogenesis. Lemon exosomes offer advantages such as high efficacy, ease of raw material acquisition, and minimal toxicity and side effects.

[0008] In a second aspect, the present invention provides the use of lemon exosomes in the preparation of a medicament for preventing, alleviating or treating diabetes.

[0009] Preferably, the lemon exosomes lower blood sugar.

[0010] The present invention found that the exosomes extracted and purified from lemons are more effective than lemon juice in lowering blood sugar.

[0011] In a third aspect, the present invention provides the use of lemon exosomes in the preparation of a medicament for promoting skin angiogenesis.

[0012] Preferably, the lemon exosomes are prepared by a method comprising the following steps:

[0013] (1) filtering the lemon juice, centrifuging the filtrate and collecting the supernatant;

[0014] (2) Filter the supernatant and perform ultracentrifugation, collect the precipitate and resuspend it.

[0015] Preferably, the centrifugation and collection of the supernatant in step (1) are performed three times in total, the first centrifugation is performed at a speed of 3000-5000xg for 5-10 min, the second centrifugation is performed at a speed of 8000-10000xg for 20-30 min, and the third centrifugation is performed at a speed of 13000-15000xg for 60-90 min.

[0016] The speed of the first centrifugation can be selected from 3000xg, 3200xg, 3400xg, 3600xg, 3800xg, 4000xg, 4200xg, 4400xg, 4600xg, 4800xg, 5000xg, etc., and the time can be selected from 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min, etc. The speed of the second centrifugation can be selected from 8000xg, 8200xg, 8400xg, 8600xg, 8800xg, 9000xg, 9200xg, 9400xg, 9600xg, 9800xg, 10000xg, etc., and the time can be selected from 20min, 21mi n, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, etc. The speed of the third centrifugation can be selected from 13000xg, 13200xg, 13400xg, 13600xg, 13800xg, 14000xg, 14200xg, 14400xg, 14600xg, 14800xg, 15000xg, etc., and the time can be selected from 60min, 62min, 65min, 68min, 70min, 72min, 75min, 78min, 80min, 82min, 85min, 88min, 90min, etc. Other specific point values within the above numerical range can be selected, so they will not be described one by one here.

[0017] Preferably, the speed of ultracentrifugation is 100,000-135,000 x g, and the time is 60-90 min.

[0018] The rotation speed can be selected as 100000xg, 102000xg, 105000xg, 108000xg, 110000xg, 112000xg, 115000xg, 118000xg, 120000xg, 122000xg, 125000xg, 128000xg, 130000xg, 132000xg, 135000xg, etc., and the time can be selected as 60min, 62min, 65min, 68min, 70min, 72min, 75min, 78min, 80min, 82min, 85min, 88min, 90min, etc. Other specific point values within the above numerical range can be selected, and they will not be repeated here.

[0019] Preferably, the dosage form of the drug includes hydrogel, tablet or granule.

[0020] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0021] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of a diluent, a dispersant, a binder, a filler, a thickener, a lubricant, a pH adjuster, a taste masking agent, a colorant, an antioxidant or an antibacterial agent.

[0022] Preferably, the skin ulcer comprises a skin ulcer caused by diabetes.

[0023] In a fourth aspect, the present invention provides the use of lemon exosomes in the preparation of macrophage inflammation inhibitors.

[0024] According to the research results of the present invention, lemon exosomes can reduce the level of inflammatory factors in vitro, that is, lemon exosomes can be made into a simple experimental preparation for reducing the level of inflammatory factors. The inflammatory inhibitor claimed by the present invention is not used to eliminate the cause or lesion of the disease, that is, it is an application in the preparation of inflammatory inhibitors for non-therapeutic purposes.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This study creatively extracts and purifies lemon exosomes and investigates their physiological effects, demonstrating their ability to reduce the expression of inflammatory factors in macrophages, promote the healing of skin ulcers, lower blood sugar, and promote skin angiogenesis. Lemon exosomes offer advantages such as high efficacy, ease of raw material acquisition, and minimal toxicity and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the flow chart for the preparation of lemon exosomes.

[0028] Figure 2 These are the test results of the in vitro biocompatibility of lemon exosome hydrogel.

[0029] Figure 3 These are the test results of the biocompatibility of lemon exosome hydrogel in vivo.

[0030] Figure 4 Lemon exosome hydrogel regulates macrophage polarization and controls inflammatory response in vitro.

[0031] Figure 5 It is the test result of the expression of pro-inflammatory M1 macrophage markers.

[0032] Figure 6 It is the test result of the expression of anti-inflammatory and pro-repair M2 macrophage markers.

[0033] Figure 7 This is a flow chart of lemon exosome hydrogel treatment of diabetic skin lesions.

[0034] Figure 8 These are the blood sugar test results before and after treatment.

[0035] Figure 9 This is a general picture of diabetic skin damage in rats before and after treatment.

[0036] Figure 10 These are HE staining images and Masson images of diabetic skin lesions before and after treatment.

[0037] Figure 11 It is the test result of skin angiogenesis. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0039] The equipment involved in the following content includes: ice box, test tube rack, ultracentrifuge tube, 50mL centrifuge tube, 1.5mL centrifuge tube, Pasteur tube, pipette tip, tweezers, 20mL syringe, Sorvall TM WX+ ultracentrifuge, freeze dryer, magnetic stirrer.

[0040] Preparation Example 1

[0041] This preparation example provides a method for preparing lemon juice, which comprises:

[0042] (1) Squeeze the lemon juice and transfer it to a 50 mL sterile centrifuge tube;

[0043] (2) The lemon juice was subjected to gradient centrifugation and the supernatant was collected (centrifugation conditions were: 5000×g for 10 min, 10000×g for 30 min, and 15000×g for 1 h) to obtain pretreated lemon juice, which was then filtered using a sterile syringe and a 0.22 μm filter to obtain sterile lemon juice.

[0044] Preparation Example 2

[0045] This preparation example provides a method for preparing lemon exosomes. The preparation flow chart is as follows: Figure 1 As shown, the specific preparation method includes:

[0046] (1) Squeeze the lemon juice and transfer it to a 50 mL sterile centrifuge tube;

[0047] (2) The lemon juice was subjected to gradient centrifugation and the supernatant was collected (centrifugation conditions were: 5000 × g for 10 min, 10000 × g for 30 min, and 15000 × g for 1 h) to obtain pretreated lemon juice, which was then filtered using a sterile syringe and a 0.22 μm filter to obtain sterile lemon juice;

[0048] (3) Exosomes were obtained from sterile lemon juice using an ultracentrifuge (ultracentrifugation conditions were: 135,000 × g for 1 h 30 min, performed under sterile conditions). After ultracentrifugation, the exosomes were enriched and finally the usable lemon exosomes were resuspended in sterile PBS.

[0049] Preparation Example 3

[0050] This preparation example provides a method for preparing a lemon exosome-loaded hydrogel, the preparation method comprising:

[0051] (1) At 80°C, 3 g of methacryloyl gelatin (GelMA) was mixed with 30 mL of PBS solution containing 0.5% (w / v) Irgacure 2959 photoinitiator to obtain a GelMA solution;

[0052] A 10% (w / v) fusarisol / sphenotype (DAS) solution was stirred in a boiling water bath until it became a paste to obtain a DAS solution;

[0053] (2) Slowly add an equal volume of DAS solution to the GelMA solution while stirring. When the temperature drops below 65°C and the gel is still in a liquid state, add an equal volume of lemon exosomes at a concentration of 20 μg / mL and mix. The resulting gel is the lemon exosome-loaded hydrogel, denoted as GelMA / DAS / Exo gel.

[0054] The lemon exosomes are the lemon exosomes obtained in Preparation Example 2.

[0055] Preparation Example 4

[0056] This preparation example provides a method for preparing a hydrogel, which comprises:

[0057] (1) At 80°C, 3 g of methacrylated gelatin (GelMA) was mixed with 30 mL of PBS solution containing 0.5% (w / v) Irgacure 2959 photoinitiator to obtain a GelMA solution;

[0058] A 10% (w / v) fusarisol / sphenotype (DAS) solution was stirred in a boiling water bath until it became a paste to obtain a DAS solution;

[0059] (2) An equal volume of DAS solution was slowly added to the GelMA solution while stirring. The resulting gel was the lemon exosome-loaded hydrogel, denoted as GelMA / DAS.

[0060] Example 1

[0061] Living-death experiments and skeleton experiments

[0062] Experimental methods:

[0063] (1) Cell live-death experiment

[0064] Fibroblast L929s cells were cultured at a rate of 1×10 6 The cells were inoculated onto the surface of the hydrogel at a density of 1000 cells / mL and co-cultured with the hydrogel in a 3 cm culture dish for 24 hours. The cells were then washed 3 times with PBS, digested with 1 mL of trypsin for 3 minutes, suspended by adding complete culture medium (10% FBS + 1% double antibody (penicillin-streptomycin) + DMEM basal culture medium), and then transferred to a 15 mL centrifuge tube and centrifuged (1000 rpm, 3 minutes) to collect the precipitate. After discarding the supernatant, the cells were washed 3 times with PBS to obtain a cell suspension. 100 μL of calcein (AM) / propidium iodide (PI) staining solution was added to 200 μL of 1×10 3 The cells were suspended in a 100 μg / mL cell suspension and then incubated at room temperature for 20 min. Cell death was observed using a confocal microscope and photographed.

[0065] (2) Cytoskeleton experiment

[0066] After 48 hours of incubation of L929s fibroblasts on the hydrogels, the culture medium was aspirated and the cells were washed three times with PBS for 3 minutes each. 4% paraformaldehyde was then added to cover the hydrogel surface and fixed on ice for 30 minutes. After cell fixation, the cells were washed three times with PBS to remove residual paraformaldehyde. Permeabilization solution was added to cover the cell surface and incubated at room temperature for 1 hour. After cell permeabilization, the cells were washed three times with PBS to remove residual permeabilization solution. 100 μL of staining solution was added to each dish and incubated in the dark for 120 minutes. The staining solution was then aspirated and the cells were washed three times with PBS for 3 minutes each. Hoechst 33342 staining solution was added. After counterstaining for 5 minutes, the cells were washed three times with PBS for 3 minutes each. Cell morphology was observed using a laser confocal microscope.

[0067] The permeabilization solution includes Solution A and Solution B. Solution A is a 6% BSA solution in PBS (600 mg BSA + 10 mL PBS), and Solution B is a 0.02% Triton solution (100 μM Triton + 5 mL PBS). Mix Solution A and Solution B in a 1:1 volume ratio to prepare the permeabilization solution.

[0068] Staining working solution: Dilute Actin-Tracker Green 488 at a ratio of 1:150 with PBS solution containing 3% BSA to prepare Actin-Tracker Green staining working solution (e.g., 10 mL PBS + 0.3 g BSA + 65 μL Actin-Tracker Green). Dilute Hochest nuclear stain at a ratio of 1:400 with antibody diluent to prepare working solution (e.g., 5 mL antibody diluent + 12.5 μL Hochest).

[0069] The results are as follows Figure 2 As shown by Figure 2 The results show that L929s cells were cultured on the hydrogel surface and live / dead staining was performed after 24 hours to determine cell viability. Live cells are represented by green, and dead cells by red. Laser confocal imaging results showed significant green fluorescence on the hydrogel surface, indicating that most cells were alive. Red fluorescence, representing dead cells, was barely visible.

[0070] Example 2

[0071] In vivo biocompatibility of lemon exosome-loaded hydrogel

[0072] Experimental methods:

[0073] Rats in the control group (untreated rats) and the experimental group (a complete skin wound (length = 10 mm) was created on the back with a scalpel, and 50 μL of 20 μg / mL GelMA / DAS / Exos hydrogel was applied to the wound surface) were sacrificed by cervical dislocation. An abdominal incision was made with a scalpel, and the skin and peritoneum were carefully dissected to expose the internal organs. The various tissues and organs were identified.

[0074] Carefully cut the aorta and vein and remove the heart intact; use a scalpel to carefully cut the liver connected to the surrounding tissue and remove the liver; carefully separate the spleen from the surrounding tissue and cut the splenic artery and vein with a scalpel and remove the spleen; carefully cut the chest cavity and remove the lungs; find the kidneys at the waist position on the back and carefully remove them.

[0075] After the organs were removed, they were fixed with 4% paraformaldehyde and the tissues were stained with HE to observe the effect of the lemon exosome hydrogel on tissue compatibility. Figure 3 As shown by Figure 3 The results showed that HE staining results showed that the lemon exosome-loaded hydrogel did not cause significant damage to organs and tissues, and there was no inflammation or necrosis in the organ tissues, proving that it had no toxic effects.

[0076] Example 3

[0077] Modulating macrophage immune inflammatory responses in vitro

[0078] Test method:

[0079] (1) The GelMA / DAS hydrogel prepared in Preparation Example 4 (GelMA / DAS group) and the injectable hydrogel loaded with lemon-derived exosomes prepared in Preparation Example 3 (GelMA / DAS / Exo group) were placed in a confocal dish and covered the bottom of the dish. After being soaked and disinfected in 75% alcohol solution for 12 hours, the culture medium was added to soak the hydrogel until the hydrogel reached full swelling equilibrium, and the culture medium was dried. L929s cells were inoculated on the surface of the hydrogel at a density of 80%, and finally the culture medium was added. The control group did not undergo any pretreatment. After the cells were inoculated, the exosomes prepared in Preparation Example 2 were added to the culture dish at an amount of 20 μg / mL for co-culture intervention.

[0080] (2) After 24 h of culture, 5 μL of LPS was added to simulate inflammation. After 12 h of stimulation, the culture medium was discarded and 1 mL of 4% paraformaldehyde was added to each dish for fixation for 30 min. The cells were then gently washed three times with PBS.

[0081] (3) Add 200 μL of pre-prepared 0.2% Triton-X to each dish to permeabilize and block the cells for 1 hour, and then gently wash with PBS three times.

[0082] (4) Add 200 μL of 10% goat serum prepared in advance to each well for blocking for 1 hour.

[0083] (5) Remove the blocking solution with a pipette, then add primary antibody (Arg-1, 1:200 / iNOS, 1:200), 200 μL per dish, and incubate at 4°C overnight.

[0084] (6) Incubate the cells overnight at 37°C for 1 h to enhance the effect of the primary antibody, and then gently wash them with PBS three times.

[0085] (7) Add 200 μL of secondary antibody goat anti-rabbit LgG Dylight 594 (1:200) or secondary antibody goat anti-rabbit LgG Dylight 488 (1:200) to each well, incubate at room temperature in the dark for 2 h, then wrap with tin foil and gently wash three times with PBS.

[0086] (8) Finally, the nuclei were stained with Hoechst 33342 for 10 min and washed three times with PBS. The expression of related proteins was observed using a laser confocal microscope.

[0087] The results are as follows Figure 4-6 As shown by Figure 4 It can be seen that the red fluorescence of ARG-1, which represents the anti-inflammatory M2 polarization, continues to increase, while the red fluorescence of iNOS, which represents the pro-inflammatory M1 polarization, continues to decrease. Figure 5 and Figure 6 It can be seen that the expression of pro-inflammatory M1 macrophage markers is reduced, while the expression of anti-inflammatory and pro-repair M2 macrophage markers is increased, proving that lemon exosomes can effectively regulate the polarization transition of macrophages, control inflammatory responses and promote tissue repair.

[0088] Example 4

[0089] Lemon exosome-loaded hydrogel specifically treats diabetic skin ulcers and prevents diabetic complications. Experimental methods:

[0090] The applicant used a full skin wound excision model to evaluate the wound healing ability of lemon exosomes in vivo. The experimental process is as follows: Figure 7 SD rats were randomly divided into four groups (blank control, GelMA / DAS, Exosomes, and GelMA / DAS / Exos). The rats were anesthetized with intraperitoneal injection of 3% sodium pentobarbital and inhaled isoflurane (2%). The backs of the rats were then shaved and disinfected.

[0091] First, in order to prove that the therapeutic effect of lemon exosomes is better than that of simple lemon water, a complete skin wound (length = 10 mm) was formed on the back with a scalpel and then divided into groups for treatment. The control group was not treated as a blood glucose control before treatment. The simple lemon water group applied 50 μL of lemon water obtained in Preparation Example 1 with a concentration of 20 μg / mL to the wound, and the gel exosome group applied 50 μL of gel exosomes obtained in Preparation Example 3 with a concentration of 20 μg / mL to the wound. The wound was then covered with a bandage to prevent the animal from scratching or biting the wound. After 14 days, the blood glucose was tested using a blood glucose meter, and the results were as follows: Figure 8 As shown in the results, both lemon water and exosomes can lower blood sugar levels, but the reduction in blood sugar after lemon exosome treatment is greater than that after lemon water. Therefore, we used lemon exosomes to load hydrogels for subsequent animal experiments.

[0092] A complete skin wound (length = 10 mm) was formed on the back with a scalpel and then divided into groups for treatment. The control group was not treated, the GelMA / DAS group applied 50 μL of the gel prepared in Preparation Example 4 to the wound, the Exosomes group applied 50 μL of the exosomes prepared in Preparation Example 2 to the wound, and the Gel / DAS / Exo group applied 50 μL of the exosome gel prepared in Preparation Example 3 to the wound. The wound was then covered with a bandage to prevent the animal from scratching or biting the wound. After recovery from anesthesia, the rats were returned to the cage and monitored every day. Digital photos of the wound were taken, and the results are shown in Figure 2. Figure 9 shown.

[0093] After 14 days, the rats were anesthetized and killed. The animal gross images and HE and Masson tissue sections showed that lemon exosome hydrogel can effectively reduce tissue inflammatory response and promote the formation of collagen fibers ( Figure 10 ); At the same time, immunohistochemistry of CD31 and VEGF in angiogenesis-related tissues proved that lemon exosome hydrogel can promote angiogenesis better than ordinary hydrogel ( Figure 11 ), proving that the exosome hydrogel has good application prospects in the treatment of diabetic skin damage.

[0094] The applicant declares that the present invention uses the above-mentioned examples to illustrate the use of lemon exosomes in the preparation of a medicament for preventing, alleviating, or treating skin ulcers. However, the present invention is not limited to the above-mentioned examples, and does not necessarily rely on the above-mentioned examples for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0095] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0096] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Use of lemon exosomes in the preparation of a medicament for preventing, alleviating or treating diabetes, wherein the lemon exosomes are prepared by a method comprising the following steps: (1) Filter the lemon juice, centrifuge the filtrate and collect the supernatant; (2) Filter the supernatant and perform ultracentrifugation, collect the precipitate and resuspend it; The centrifugation and collection of the supernatant in step (1) are performed three times in total, with the first centrifugation being performed at a speed of 3000-5000 xg for 5-10 min; the second centrifugation being performed at a speed of 8000-10000 xg for 20-30 min; and the third centrifugation being performed at a speed of 13000-15000 xg for 60-90 min. The ultracentrifugation speed is 100,000-135,000 x g and the time is 60-90 min.

2. The use according to claim 1, characterized in that The dosage form of the drug includes hydrogel, tablet or granule.

3. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that The pharmaceutically acceptable excipients include any one or a combination of at least two of diluents, dispersants, binders, fillers, thickeners, lubricants, pH regulators, taste masking agents, colorants, antioxidants or antibacterial agents.

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