Application of lemon exosome in preparation of medicine for preventing, relieving or treating skin ulcer

Abstract

The invention relates to application of lemon exosomes in preparation of medicines for preventing, relieving or treating skin ulcers, exosomes in lemons are creatively extracted and purified, the physiological effects of the exosomes are researched, the lemon exosomes can reduce the expression level of inflammatory factors in macrophages and can promote healing of the skin ulcers, and the application of the lemon exosomes in preparation of medicines for preventing, relieving or treating the skin ulcers is achieved. Blood sugar is reduced, and skin and blood vessel regeneration is promoted. The lemon exosome has the advantages of good curative effect, simple raw material acquisition and small toxic and side effects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application 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 "thirstiness", "sores", and "ulcers". The causes are divided into internal and external factors. The internal factors mainly include "emotional injuries, improper diet, and sexual injury", and the external factors mainly include "six evil toxins and special poisons". The pathogenesis is mainly attributed to "stagnation of qi and blood, obstruction of meridians, stasis and damage to the body, and damage caused by corruption". At present, conventional treatments for diabetic wounds include controlling blood sugar, plant transplantation, surgical debridement, and adding 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 have become an emerging frontier in therapeutic and targeted drug delivery. Plant-derived extracellular vesicles have shown their unique advantages, such as safety, great possibilities for large-scale preparation, 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 an application 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 the use of lemon exosomes in the preparation of a medicament for preventing, alleviating or treating skin ulcers.

[0007] The present invention creatively extracts and purifies exosomes from lemons and studies their physiological effects, which can reduce the expression level 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 low 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 finds 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 3000-5000 x g for 5-10 min, the second centrifugation is performed at a speed of 8000-10000 x g for 20-30 min, and the third centrifugation is performed at a speed of 13000-15000 x g 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 The speed of the third centrifugation can be selected as 13000xg, 13200xg, 13400xg, 13600xg, 13800xg, 14000xg, 14200xg, 14400xg, 14600xg, 14800xg, 15000xg, 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, so they will not be described one by one here.

[0017] Preferably, the speed of ultracentrifugation is 100000-135000 xg, 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, which will not be repeated here.

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

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

[0021] Preferably, the pharmaceutically acceptable excipients include any one 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, or a combination of at least two thereof.

[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, 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] The present invention creatively extracts and purifies exosomes from lemons, and studies their physiological effects, which can reduce the expression level 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 low toxicity and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart of 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 hydrogels regulate macrophage polarization in vitro to control inflammatory responses.

[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 in the treatment of diabetic skin lesions.

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

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

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

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

[0038] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only 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, gun 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, the preparation method comprising:

[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 (the centrifugation conditions were: 5000 x g for 10 min, 10000 x g for 30 min, and 15000 x 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) subjecting the lemon juice to gradient centrifugation and collecting the supernatant (the centrifugation conditions are: 5000 x g for 10 min, 10000 x g for 30 min, and 15000 x g for 1 h) to obtain pretreated lemon juice, and then filtering the juice using a sterile syringe and a 0.22 μm filter to obtain sterile lemon juice;

[0048] (3) Using an ultracentrifuge to obtain exosomes from sterile lemon juice (the ultracentrifugation conditions are: 135,000 x g for 1 h 30 min, performed under sterile conditions), enriching the exosomes after ultracentrifugation, and finally resuspending the obtained usable lemon exosomes in sterile PBS.

[0049] Preparation Example 3

[0050] This preparation example provides a method for preparing a hydrogel loaded with lemon exosomes, 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) 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 20 μg / mL lemon exosomes and mix. The resulting gel is a hydrogel loaded with lemon exosomes, recorded 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, the preparation method comprising:

[0057] (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;

[0058] A 10% (w / v) 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 were cultured at 1×10 6 The cells were inoculated on the surface of the hydrogel at a density of 100 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 for centrifugation (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 then incubated at room temperature for 20 min. Confocal microscopy was used to observe cell death and take photos.

[0065] (2) Cytoskeleton experiments

[0066] After the hydrogel and fibroblast L929s cells were cultured for 48 hours, the culture medium was aspirated and washed with PBS three times for 3 minutes each time. Then 4% paraformaldehyde was added to cover the surface of the hydrogel and fixed on ice for 30 minutes. After the cell fixation was completed, it was washed with PBS three times to remove the residual paraformaldehyde. The membrane permeabilization solution was added to cover the cell surface and incubated at room temperature for 1 hour. After the cell membrane was permeabilized, it was washed with PBS three times to remove the residual membrane permeabilization solution, and 100 μL of the staining working solution was added to each dish and incubated in the dark for 120 minutes. Then the staining working solution was aspirated and washed with PBS three times for 3 minutes each time. Hoechst 33342 staining solution was added. After re-staining for 5 minutes, it was washed with PBS three times for 3 minutes each time. The cell morphology was observed using a laser confocal microscope.

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

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

[0069] The results are as follows Figure 2 As shown by Figure 2 The results show that L929s cells were cultured on the surface of the hydrogel material, and live / dead staining was performed after 24 hours of culture to determine the viability of the cells. Live cells are represented by green, and dead cells are represented by red; laser confocal imaging results show that significant green fluorescence is observed on the surface of the hydrogel, indicating that most cells are alive. The red fluorescence representing dead cells is almost invisible.

[0070] Example 2

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

[0072] Experimental methods:

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

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

[0075] After the above 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 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 response 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 so that the hydrogel fully reached 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 perform any pretreatment. After the cells were inoculated, the exosomes group (Exosomes) added the exosomes prepared in Preparation Example 2 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 dishes were then gently washed three times with PBS.

[0081] (3) Add 200 μL of 0.2% Triton-X prepared in advance 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, 200 μL per dish (Arg-1, 1:200 / iNOS, 1:200), 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 three times with PBS.

[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-repairing M2 macrophage markers is increased, proving that lemon exosomes can effectively regulate the polarization transition of macrophages, control the inflammatory response 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 group, GelMA / DAS group, Exosomes group, GelMA / DAS / Exos group), and the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital and inhalation of isoflurane (2%). The hair on the back of the rats was 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 sugar 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. Then cover the wound with a bandage to prevent the animal from scratching or biting the wound. After 14 days, the blood sugar was tested using a blood glucose meter, and the results are as follows: Figure 8 As shown in the results, both lemon water and exosomes can reduce blood sugar levels, but the decrease 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 was smeared with 50 μL of the gel prepared in Preparation Example 4 on the wound, the Exosomes group was smeared with 50 μL of the exosomes prepared in Preparation Example 2 on the wound, and the Gel / DAS / Exo group was smeared with 50 μL of the exosome gel prepared in Preparation Example 3 on the wound. The wound was then covered with a bandage to prevent the animal from scratching or biting the wound. After anesthesia was recovered, 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. Fig. 9 shown.

[0093] After 14 days, the rats were anesthetized and killed. The animal gross images and HE and Masson tissue sections proved that lemon exosome hydrogel can effectively reduce tissue inflammatory response and promote the formation of collagen fibers ( Fig.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 ( Fig.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 embodiments to illustrate the application of the lemon exosomes of the present invention in the preparation of drugs for preventing, alleviating or treating skin ulcers, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope 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 suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Application of lemon exosomes in the preparation of drugs for preventing, alleviating or treating skin ulcers.

2. Application of lemon exosomes in the preparation of drugs for preventing, alleviating or treating diabetes.

3. Application of lemon exosomes in the preparation of drugs for promoting skin angiogenesis.

4. The use according to any one of claims 1 to 3, characterized in that: The lemon exosomes are prepared by a method comprising the following steps: (1) filtering the lemon juice, centrifuging the filtrate and collecting the supernatant; (2) Filter the supernatant and perform ultracentrifugation, collect the precipitate and resuspend it.

5. The use according to claim 4, characterized in that: 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 3000-5000 x g for 5-10 min; the second centrifugation is performed at a speed of 8000-10000 x g for 20-30 min; the third centrifugation is performed at a speed of 13000-15000 x g for 60-90 min.

6. The use according to claim 4 or 5, characterized in that: The speed of ultracentrifugation is 100000-135000xg, and the time is 60-90min.

7. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug includes hydrogel, tablet or granule.

8. The use according to any one of claims 1 to 7, characterized in that: The drug also includes pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include any one 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, or a combination of at least two thereof.

9. The use according to any one of claims 1 to 8, characterized in that: The skin ulcers include skin ulcers caused by diabetes.

10. Application of lemon exosomes in the preparation of macrophage inflammation inhibitors.

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