Application of Euphorbia humifusa - derived extracellular vesicles in the preparation of drugs for preventing and treating ulcerative colitis

Abstract

The present invention belongs to the field of treatment of ulcerative colitis and provides an application of extracellular vesicles derived from Herba Euphorbiae in preparing a drug for preventing and treating ulcerative colitis. The application includes extraction and preparation of extracellular vesicles derived from Herba Euphorbiae and research on the preventive and therapeutic effects of extracellular vesicles derived from Herba Euphorbiae on ulcerative colitis in mice. The specific steps are as follows: Step 1, extracting leaf cells from Herba Euphorbiae: selecting fresh and healthy Herba Euphorbiae leaves as experimental materials and chopping them into pieces, adding the chopped samples into an enzymatic hydrolysis solution, ensuring that the samples are fully in contact with the enzymatic hydrolysis solution and incubating them, gently turning or stirring during the incubation to promote the enzymatic hydrolysis process, thereby isolating the leaf cells; Step 2, culturing the leaf cells of Herba Euphorbiae: culturing the leaf cells of Herba Euphorbiae in a culture medium; The present invention confirms that extracellular vesicles derived from Herba Euphorbiae can be used to treat ulcerative colitis, provides an experimental basis for the development of new drugs for the clinical prevention and treatment of IBD, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of ulcerative colitis treatment, and specifically is an application of extracellular vesicles derived from Euphorbia pulex in the preparation of a medicine for preventing and treating ulcerative colitis. Background Art

[0002] Ulcerative colitis is an inflammatory bowel disease characterized by chronic, nonspecific inflammation of the colon and rectum, with lesions typically confined to the large intestinal mucosa and submucosa. Currently, the first-line treatment for inducing and maintaining remission in mild to moderate ulcerative colitis is 5-aminosalicylic acid, while moderate to severe ulcerative colitis requires oral corticosteroids for induction of remission. In addition, treatments targeting specific inflammatory pathways include monoclonal antibodies against tumor necrosis factor, α4β7 integrin, IL-12, and IL-23 cytokines, and oral small molecule therapies targeting janus kinase or sphingosine-1-phosphate. While these treatments can alleviate UC symptoms, they are associated with uncertain efficacy, high drug dependency, the development of drug resistance, and side effects. Therefore, patients with UC urgently need new, safe, effective, and cost-effective treatments.

[0003] With the advancement of nanomedicine, numerous synthetic nanoparticle therapeutics have been developed for specific drug delivery to the colon. However, their preparation processes are complex and costly, production scale is limited, stability and consistency are difficult to ensure, and there are potential biocompatibility and toxicity issues. In contrast, compared to traditional synthetic nanoparticles prepared in a chemical environment, extracellular vesicles (EVs) derived from edible plants have the advantages of low immunogenicity, high biocompatibility and bioavailability, and simple preparation. These advantages make plant-derived EVs highly promising for clinical applications in the treatment of various diseases, including UC.

[0004] Extracellular vesicle is the nanoscale or micron-sized membranous vesicle released by cells, can carry the bioactive molecules such as protein, lipid, nucleic acid, and plays an important role in intercellular communication and material exchange. The extracellular vesicle derived from Herba Euphorbiae is considered to contain multiple pharmacologically active ingredients, and these active ingredients may participate in inflammation-related signaling pathways and biological functions by acting on specific target sites (such as AKT1, PPARG and IL6 etc.). But the current scientific research to it is still insufficient, and its abundant pharmacological value and application prospects cannot be fully and deeply excavated, and the extracellular vesicle in Herba Euphorbiaee source is not yet seen to be used in the prevention and treatment research of ulcerative colitis. For this reason, those skilled in the art have proposed a kind of Herba Euphorbiaee source extracellular vesicle application in the preparation of prevention and treatment ulcerative colitis medicine to solve the problem that background technology proposes. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an application of extracellular vesicles derived from Euphorbia pulex in the preparation of drugs for preventing and treating ulcerative colitis, so as to solve the problems in the existing technology of treating ulcerative colitis such as unclear efficacy, strong drug dependence, easy drug resistance and side effects.

[0006] A use of extracellular vesicles derived from Euphorbia pulex in preparing a drug for preventing and treating ulcerative colitis, comprising:

[0007] The extraction and preparation of extracellular vesicles derived from Herba Euphorbiae and the study of the preventive and therapeutic effects of extracellular vesicles derived from Herba Euphorbiaee on ulcerative colitis in mice are as follows:

[0008] Step 1: Extraction of leaf cells from Euphorbia pulex: Select fresh, healthy Euphorbia pulex leaves as experimental materials and chop them into small pieces. Add the chopped sample to the enzymatic hydrolysis solution, ensuring full contact between the sample and the solution and incubating. Gently flip or stir the sample during incubation to promote the enzymatic hydrolysis process, thereby isolating the leaf cells.

[0009] Step 2, culturing leaf cells of Herba Euphorbiae Herba: culturing leaf cells of Herba Euphorbiae Herba in a culture medium, so that the leaf cells grow and reproduce normally and secrete extracellular vesicles;

[0010] Step 3: Isolation and purification of extracellular vesicles: Leaf cells secrete extracellular vesicles during culture. These vesicles are isolated and purified by ultracentrifugation and then characterized to determine their size, morphology, and surface markers.

[0011] Step 4: Collect and extract the extracellular vesicle fluid and store it in a -20°C refrigerator for future use;

[0012] Step 5, mouse grouping and drug administration: 18 6-8 week old C57BL / 6 mice were randomly divided into three groups, with 6 mice in each group, namely: blank group (Control), DSS group (3% DSS water) and DSS + Herba Euphorbiae Herba-derived extracellular vesicles group (DSS + EHH-ExoNPs). The mice in the blank group had free access to drinking water, and the other two groups of mice had 3% (w / w) DSS added to their drinking water and drank freely for 7 days. The concentration of Herba Euphorbiae-derived extracellular vesicles was adjusted to 10 mg / ml. During the DSS modeling period, the intervention group was continuously gavaged for 7 days with a gavage volume of 200 μl / mouse; the DSS group was gavaged with an equal amount of phosphate buffered saline (PBS);

[0013] Step 6. Throughout the experiment, the body weight, fecal characteristics, and physical activity of the three groups of mice were monitored daily, and the disease activity index (DAI) scores were calculated. The data were collated for comparison. On the seventh day, the mice were euthanized using CO2 inhalation, and the colon and rectal tissues were collected, and the colon length was measured for further experiments.

[0014] Preferably, the enzymatic hydrolysis temperature for separating leaf cells is set to 45°C, the enzymatic hydrolysis pH is 4.5, the enzymatic hydrolysis time is 45 minutes, and the enzymatic hydrolysis solution is a mixed enzymatic hydrolysis solution of 0.6% cellulase and 0.25% pectinase. The leaf cells are released after degrading the plant cell wall. During the enzymatic hydrolysis process, the pH value is maintained stable by the buffer solution to protect the leaf cells from damage.

[0015] Preferably, leaf cells are cultured under sterile conditions using MS as the basic culture medium, cytokinins, production factors and 2,4-D growth regulators are added, the pH value of the MS culture medium is adjusted to 5.8-6.0, the culture temperature is 20-28°C, the sucrose concentration is 30g / L, and during the leaf cell culture period, the light duration is 12h, alternating with dark time, and the illumination is 2500Lx.

[0016] Preferably, the mixture after enzymatic hydrolysis is centrifuged step by step at a speed of 300-3500×g to remove undigested cell debris and impurities, and the supernatant after centrifugation (containing extracellular vesicles) is collected and ultracentrifuged at a speed of 100,000×g. The supernatant is discarded, and the extracellular vesicles are resuspended with phosphate buffer. The resuspended extracellular vesicles are added to an ultracentrifuge tube containing 60 wt%, 45 wt%, and 30 wt% sucrose solutions, and then ultracentrifuged at 140,000×g at 4°C for 60-90 min. The liquid band between 45 wt% and 30 wt% is aspirated and mixed evenly with PBS. The sucrose solution is removed by further ultracentrifugation at 150,000×g at 4°C for 60-90 min. The resulting precipitate is resuspended with PBS to obtain extracellular vesicles derived from Herba Euphorbiae.

[0017] Preferably, the changes in the percentage of weight loss of mice in the normal group, DSS model group, and EHH-ExoNPs intervention group are observed and recorded over a period of 7 days.

[0018] Preferably, the disease activity index (DAI) scores of the normal group mice, the DSS model group, and the EHH-ExoNPs intervention group mice are observed and recorded every day during the experimental period. The evaluation contents include the percentage of weight loss of the mice, stool viscosity, and stool bleeding.

[0019] Preferably, on the 7th day at the end of the experiment, the mice were euthanized by CO2 inhalation, and the colons of the normal group mice, the DSS model group and the EHH-ExoNPs intervention group mice were collected and their lengths were measured.

[0020] Preferably, on the seventh day after the end of the experiment, the rectal part of the mouse 0.5 cm away from the anus is taken, routinely dehydrated and embedded, and then subjected to hematoxylin-eosin (HE) staining, immunohistochemical staining, and glycogen staining to observe the pathological damage of the mouse colon, the expression of E-Cadherin, and the number of goblet cells.

[0021] Preferably, RNA is extracted from mouse colon samples using TRI zo l reagent, the RNA concentration is determined and reverse transcribed, and the relative expression levels of IL-6, IL-1β and TNF-α proinflammatory cytokines in normal group mice, DSS model group and EHH-ExoNPs intervention group mice are detected by qPCR.

[0022] Preferably, the qPCR method is used to detect the relative expression levels of protective barrier-related factors Occludin and Claudin in the normal group mice, DSS model group and EHH-ExoNPs intervention group mice.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. This invention uses enzymatic hydrolysis combined with leaf cell culture to extract extracellular vesicles from Euphorbia milii. Compared to conventional grinding and crushing to extract extracellular vesicles, enzymatic hydrolysis degrades the cell walls and intercellular connective materials of leaf cells, making it easier to release cellular contents, thereby improving the extraction efficiency of extracellular vesicles. Furthermore, the leaf cell reproduction process can further increase the cell number, thereby increasing the yield of extracellular vesicles.

[0025] 2. The present invention is the first to use extracellular vesicles derived from Herba Dichroae for the treatment of ulcerative colitis in mice. A mouse colitis model was induced using 3% DSS, and the mice were divided into a normal group, a DSS model group, and an EHH-ExoNPs treatment group. Animal experiments were conducted on the three groups of mice and compared. The experimental results showed that compared with the mice in the DSS model group, the mice in the EHH-ExoNPs intervention group had a lower percentage of weight loss, diarrhea and bloody stools, and a lower DAI score. In addition, the shortening of the colon length of the mice in the EHH-ExoNPs intervention group was alleviated, and their colon length was longer than that of the mice in the DSS model group. H&E section staining and Alcian blue-periodic acid Schiff staining (AB-PAS) staining results showed that the colon of mice with DSS-induced colitis was characterized by large-scale and extensive epithelial cell attenuation or erosion, goblet cell depletion, large areas of no crypts, inflammatory cell infiltration of the lamina propria, and thickening of the submucosal layer. The pathological phenotype was the same as that of patients with ulcerative colitis. Treatment with extracellular vesicles derived from Euphorbia humulosa (EHH-ExoNPs) significantly alleviated DSS-induced pathological damage; crypt glands were observed; inflammatory infiltration was concentrated in the lamina propria; there was no significant mixed infiltration, fewer damaged epithelial cells, and more goblet cells than in the DSS group. Immunohistochemistry revealed that E-Cadherin expression was significantly increased in the EHH-ExoNPs-treated group compared with the DSS colitis model group. qPCR revealed that compared with DSS-induced colitis mice, the relative mRNA expression levels of the proinflammatory cytokines IL-6, TNF-α, and IL-1β in the intervention group were significantly decreased, while the expression levels of the protective barrier-associated factors Occludin and Claudin were increased.

[0026] These experimental results demonstrate that extracellular vesicles derived from Herba Euphorbiae Herba can effectively treat and alleviate symptoms of ulcerative colitis, including weight loss, diarrhea, and bloody stools. They can also reduce the levels of inflammatory factors in the colonic tissue of colitis mice and alleviate the DSS-induced inflammatory response. Furthermore, they can increase mucus production and E-cadherin expression in intestinal epithelial goblet cells, further protecting the mucosal barrier and repairing colonic damage. Therefore, extracellular vesicles derived from Herba Euphorbiae Herba can be an effective treatment for ulcerative colitis.

[0027] In summary, the present invention confirms that extracellular vesicles derived from Euphorbia pulex can be used to prevent or treat ulcerative colitis, provides a new theoretical basis and experimental foundation for the development of new drugs for the clinical prevention and treatment of UC, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a transmission electron micrograph of the extracellular vesicles of Herba Euphorbiae rutaecarpa of the present invention;

[0029] Figure 2 This is a distribution diagram of the particle size range of the extracellular vesicles of Herba Euphorbiae rutaecarpa of the present invention;

[0030] Figure 3 The figure is a distribution diagram of particle size and concentration of extracellular vesicles of Euphorbia pulex measured by nanoparticle tracking analysis (NTA) in the present invention;

[0031] Figure 4 Figure 2 is a graph showing the percentage of weight loss in the normal group, DSS model group, and EHH-ExoNPs intervention group of mice in the present invention (n=6; *, P<0.05; **, P<0.01; ***, P<0.001, mean±SEM);

[0032] Figure 5 The disease activity index (DAI) score diagram of the normal group mice, DSS model group and EHH-ExoNPs intervention group mice of the present invention (n=6; *, P<0.05; **, P<0.01; ***, P<0.001, mean±SEM);

[0033] Figure 6 、 7 Figure 2 shows the actual colon length of mice in the normal group, DSS model group, and EHH-ExoNPs intervention group (n=6; *, P<0.05; **, P<0.01; ***, P<0.001, mean±SEM).

[0034] Figure 8 Figure 3 is the hematoxylin-eosin (HE) staining, immunohistochemical staining, and Alcian blue-periodic acid Schiff (AB-PAS) staining of mice in the normal group, DSS model group, and EHH-ExoNPs intervention group of the present invention;

[0035] Figure 9 Statistical graph of the goblet cell numbers of mice in the normal group, DSS model group, and EHH-ExoNPs intervention group of the present invention (n=6; *, P<0.05; **, P<0.01; ***, P<0.001, mean±SEM);

[0036] Figure 10 、 11 , 12 are the relative mRNA expressions of related pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the DSS model group and EHH-ExoNPs intervention group of the present invention (n=6; *, P<0.05; **, P<0.01; ***, P<0.001, mean±SEM);

[0037] Figure 13 、 14The graph shows the relative mRNA expression of protective barrier-related factors Occludin and Cludin in the DSS model group and EHH-ExoNPs intervention group of the present invention (n=6; *, P<0.05; **, P<0.01; ***, P<0.001, mean±SEM). DETAILED DESCRIPTION

[0038] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0039] Reference Figures 1-14 The present invention provides an application of extracellular vesicles derived from Euphorbia pulex in preparing a drug for preventing and treating ulcerative colitis, comprising:

[0040] The extraction and preparation of extracellular vesicles derived from Herba Euphorbiae and the study of the preventive and therapeutic effects of extracellular vesicles derived from Herba Euphorbiaee on ulcerative colitis in mice are as follows:

[0041] Step 1: Extraction of leaf cells from Euphorbia pulex: Select fresh, healthy Euphorbia pulex leaves as experimental materials and chop them into small pieces. Add the chopped sample to the enzymatic hydrolysis solution, ensuring full contact between the sample and the solution and incubating. Gently flip or stir the sample during incubation to promote the enzymatic hydrolysis process, thereby isolating the leaf cells.

[0042] Step 2, culturing leaf cells of Herba Euphorbiae Herba: culturing leaf cells of Herba Euphorbiae Herba in a culture medium, so that the leaf cells grow and reproduce normally and secrete extracellular vesicles;

[0043] Step 3: Isolation and purification of extracellular vesicles: Leaf cells secrete extracellular vesicles during culture. These vesicles are isolated and purified by ultracentrifugation and then characterized to determine their size, morphology, and surface markers.

[0044] Step 4: Collect and extract the extracellular vesicle fluid and store it in a -20°C refrigerator for future use;

[0045] Step 5, mouse grouping and drug administration: 18 6-8 week old C57BL / 6 mice were randomly divided into three groups, with 6 mice in each group, namely: blank group (Control), DSS group (3% DSS water) and DSS + Herba Euphorbiae Herba-derived extracellular vesicles group (DSS + EHH-ExoNPs). The mice in the blank group had free access to drinking water, and the other two groups of mice had 3% (w / w) DSS added to their drinking water and drank freely for 7 days. The concentration of Herba Euphorbiae-derived extracellular vesicles was adjusted to 10 mg / ml. During the DSS modeling period, the intervention group was continuously gavaged for 7 days with a gavage volume of 200 μl / mouse; the DSS group was gavaged with an equal amount of phosphate buffered saline (PBS);

[0046] Step 6. Throughout the experiment, the body weight, fecal characteristics, and physical activity of the three groups of mice were monitored daily, and the disease activity index (DAI) scores were calculated. The data were collated for comparison. On the seventh day, the mice were euthanized using CO2 inhalation, and the colon and rectal tissues were collected, and the colon length was measured for further experiments.

[0047] As can be seen from the above, (1) extracellular vesicles derived from Herba Euphorbiae Herba were extracted and prepared by enzymatic hydrolysis and leaf cell culture methods; (2) extracellular vesicles derived from Herba Euphorbiae Herba were used in animal experiments: a 3% DSS-induced mouse colitis model was used, and extracellular vesicles derived from Herba Euphorbiae Herba (EHH-ExoNPs) were orally fed to colitis mice to observe its alleviating and therapeutic effects on mouse colitis.

[0048] Specifically, the enzymatic hydrolysis temperature for separating leaf cells is set to 45°C, the enzymatic hydrolysis pH is 4.5, the enzymatic hydrolysis time is 45 minutes, and the enzymatic hydrolysis solution is a mixed enzymatic hydrolysis solution of 0.6% cellulase and 0.25% pectinase. After degrading the plant cell wall, the leaf cells are released. During the enzymatic hydrolysis process, the pH value is maintained stable by a buffer solution to protect the leaf cells from damage.

[0049] From the above, it can be seen that after setting appropriate enzymatic hydrolysis parameters and adjustments, the leaf cells in Euphorbia pulegium can be separated by a mixed enzymatic hydrolysis solution of 0.6% cellulase and 0.25% pectinase.

[0050] Specifically, leaf cells were cultured under sterile conditions using MS as the basic culture medium, cytokinins, production factors and 2,4-D growth regulators were added, the pH value of the MS culture medium was adjusted to 5.8-6.0, the culture temperature was 20-28°C, the sucrose concentration was 30 g / L, and during the leaf cell culture period, the light duration was 12 hours, alternating with dark time, and the illumination was 2500 Lx.

[0051] From the above, we can see that by culturing leaf cells in MS medium and setting reasonable parameter conditions, the normal growth and reproduction of leaf cells can be maintained.

[0052] Specifically, the mixture after enzymatic hydrolysis is centrifuged step by step at a speed of 300-3500×g to remove undigested cell debris and impurities, the supernatant after centrifugation (containing extracellular vesicles) is collected and ultracentrifuged at a speed of 100,000×g, the supernatant is discarded, the extracellular vesicles are resuspended with phosphate buffer, and the resuspended extracellular vesicles are added to an ultracentrifuge tube containing 60 wt%, 45 wt%, and 30 wt% sucrose solutions, and then ultracentrifuged at 140,000×g for 60-90 min at 4°C, the liquid band between 45 wt% and 30 wt% is aspirated, mixed evenly with PBS, and continued ultracentrifuged at 150,000×g for 60-90 min at 4°C to remove the sucrose solution, and the resulting precipitate is resuspended with PBS to obtain extracellular vesicles derived from Herba Euphorbiae.

[0053] As can be seen from the above, extracellular vesicles of Herba Euphorbiae were obtained by ultracentrifugation, and the extracellular vesicles were resuspended in a buffer solution and aliquoted and frozen at -20°C. Figure 1 、 2 As shown in Figures 3 and 4, the extracellular vesicles extracted and prepared from Herba Euphorbiae are small, round, transparent vesicles with a particle size distribution range of 60-240 nanometers.

[0054] Specifically, starting from the second day of modeling, the changes in the percentage of weight loss of mice in the normal group, DSS model group, and EHH-ExoNPs intervention group were observed and recorded daily.

[0055] From the above, we can see that by observing the weight changes of the three groups of mice, we can detect whether the extracellular vesicles derived from Herba Euphorbiae Herba have the effect of alleviating weight loss in DSS-induced colitis mice. Figure 4 As shown in the results, compared with the stable weight growth of mice in the normal group, the weight of mice in the DSS colitis model group decreased sharply, and by the seventh day their weight had dropped to about 70% of their initial weight (Control: DSS, P < 0.0001). In contrast, the weight loss of mice in the treatment group fed with extracellular vesicles of Euphorbia cerevisiae was alleviated (EHH-ExoNPs: DSS, P < 0.05).

[0056] Specifically, the disease activity index (DAI) scores of mice in the normal group, DSS model group, and EHH-ExoNPs intervention group were observed and recorded daily during the experimental period. The assessment included the percentage of weight loss, stool consistency, and stool bleeding. The specific scoring criteria are as follows: The disease activity index combines the percentage of weight loss (no weight loss = 0, 1-5% = 1 point, 5-10% = 2 points, 10-20% = 3 points, and more than 20% = 4 points), stool consistency (normal = 0, loose stool = 2 points, diarrhea = 4 points), and stool bleeding (normal = 0, positive occult blood = 2 points, and overt bleeding = 4 points) to form a comprehensive score. The DAI value is obtained by adding the scores of the three results and dividing by 3. That is, DAI = (body weight index + stool shape + bleeding) / 3.

[0057] From the above, we can know that the DAI score of mice is obtained by the percentage of weight loss, stool viscosity and stool bleeding of mice, so as to compare the health status of the three groups of mice and make a DAI score line graph, as shown in the figure below. Figure 5 As shown in the figure, compared with the normal group mice, the DAI score of the DSS colitis mice was higher (Control: DSS, P < 0.0001), while the DAI score of the mice treated with extracellular vesicles of Euphorbia cerevisiae was reduced (EHH-ExoNPs: DSS, P < 0.01).

[0058] Specifically, on the 7th day after the end of the experiment, the mice were euthanized by CO2 inhalation, and the colons of the mice in the normal group, DSS model group, and EHH-ExoNPs intervention group were collected and their lengths were measured. Figure 6 、 7 As shown in the data, compared with the normal group mice, the colon length of the DSS colitis mice was shorter (Control: DSS, P < 0.0001), while the shortening of the colon length was alleviated in the mice treated with extracellular vesicles of Euphorbia pulex (EHH-ExoNPs: DSS, P < 0.01).

[0059] Specifically, on the seventh day after the end of the experiment, the rectal area 0.5 cm away from the anus of the mouse was taken, routinely dehydrated and embedded, and then subjected to hematoxylin-eosin (HE) staining, immunohistochemistry staining, and glycogen staining to observe the pathological damage of the mouse colon, the expression of E-Cadherin, and the number of goblet cells.

[0060] The specific implementation is as follows: After reaching the end point of the experiment, the mice were euthanized by CO2 inhalation. The rectal tissue 0.5 cm long from the anus of each group of mice was fixed in 4% paraformaldehyde at room temperature for 24 hours, and then placed in a tissue dehydrator for dehydration overnight. The tissue was then immersed in wax and embedded. The tissue was sliced at a thickness of 4 μm on a paraffin microtome, baked at 75°C for 1.5 hours, placed on a slide to air dry, and stored at room temperature for later use.

[0061] Hematoxylin-eosin (HE) staining:

[0062] 1) Dewaxing of paraffin sections: Place sections in environmentally friendly dewaxing solution for 30 minutes, then in anhydrous ethanol I for 5 minutes, then in anhydrous ethanol II for 5 minutes, then in 95% ethanol for 5 minutes, then in 90% ethanol for 5 minutes, then in 80% ethanol for 5 minutes, then in 70% ethanol for 5 minutes, and finally in distilled water.

[0063] 2) Hematoxylin staining of cell nuclei: Stain sections with Harris hematoxylin for 3-8 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, blue with 0.6% ammonia solution, and rinse with running water.

[0064] 3) Eosin staining of cytoplasm: Slice into eosin staining solution and stain for 1-3 minutes.

[0065] 4) Dehydration and Mounting: Dehydrate the sections in 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and environmentally friendly dewaxing solution for 30 min. Mount the sections with neutral gum and observe under an optical microscope and take photos.

[0066] Immunohistochemical staining:

[0067] 1) Dewaxing of paraffin sections: Place sections in environmentally friendly dewaxing solution for 30 minutes, then in anhydrous ethanol I for 5 minutes, then in anhydrous ethanol II for 5 minutes, then in 95% ethanol for 5 minutes, then in 90% ethanol for 5 minutes, then in 80% ethanol for 5 minutes, then in 70% ethanol for 5 minutes, and finally in distilled water for 3 minutes.

[0068] 2) Inactivation of endogenous enzymes: Soak the sections in 3% hydrogen peroxide for 5 minutes, and then soak in PBS for 2 minutes.

[0069] 3) Antigen retrieval: Add EDTA retrieval solution to the retrieval box, place the slide in the retrieval box, place the retrieval box in a 100°C water bath for 20 minutes or in a 120°C autoclave for 2-3 minutes, cool naturally, and finally soak in PBS for 2 minutes.

[0070] 4) Blocking and Antibody Incubation: Draw a water-blocking circle on a slide, add a drop of diluted E-cadherin primary antibody to cover the tissue, and place in a moisturizing box at 4°C overnight. After overnight, soak the sections in PBS three times for 3 minutes each to remove unbound primary antibody. Add a drop of diluted secondary antibody to cover the tissue, and place in a moisturizing box at room temperature for 20 minutes. Soak the sections in PBS three times for 3 minutes each to remove unbound secondary antibody.

[0071] 5) Color development: Add DAB working solution (freshly prepared for use) to cover the tissue, stain for 5-15 minutes, and rinse the sections with purified water to stop the color development reaction.

[0072] 6) Counterstaining and Mounting: Add hematoxylin to cover the tissue for counterstaining, staining the cell nuclei blue. Rinse the sections in purified water for 3 minutes. After dehydration with a gradient of alcohol and soaking in a clearing solution, remove the slides and air dry them. Apply a drop of neutral resin and cover with a coverslip. Gently press the coverslip to remove air bubbles and preserve the staining effect.

[0073] Alcian blue-periodic acid Schiff stain (AB-PAS) staining:

[0074] 1) The tissue was fixed with Canon's fixative and the sections were dewaxed as above;

[0075] 2) Stain with Alcian blue solution for 5-10 minutes and then rinse with water; stain with periodic acid for 5 minutes and then rinse with running water for several minutes; stain with Schiff's reagent in the dark for 5-10 minutes and then rinse with running water for 5-10 minutes; stain the nucleus with hematoxylin for 1 minute and then rinse with running water.

[0076] 3) Dehydration, transparency, sealing, and microscopic examination (same as immunohistochemistry).

[0077] From the above, we can see that Figure 8 、 9 H&E section staining and Alcian blue-periodic acid-Schiff (AB-PAS) staining revealed that the colons of mice with DSS-induced colitis were characterized by extensive epithelial cell attenuation or erosion, goblet cell depletion (Control:DSS, P<0.0001), large areas of crypt absence, inflammatory cell infiltration of the lamina propria, and thickening of the submucosal layer. Treatment with extracellular vesicles derived from Euphorbia miliariae (EHH-ExoNPs) significantly alleviated DSS-induced pathological damage, with crypt glands observed and inflammatory infiltration concentrated in the lamina propria. There was no significant mixed infiltration, fewer damaged epithelial cells, and more goblet cells compared to the DSS group (EHH-ExoNPs:DSS, P<0.001). Immunohistochemistry revealed significantly increased E-Cadherin expression in the EHH-ExoNPs-treated group compared to the DSS colitis model group.

[0078] Specifically, TRI zo l reagent was used to extract RNA from mouse colon samples, determine the RNA concentration and perform reverse transcription, and qPCR was used to detect the relative expression levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α in normal group mice, DSS model group and EHH-ExoNPs intervention group mice.

[0079] Specifically, the qPCR method was used to detect the relative expression levels of protective barrier-related factors Occludin and Claudin in the normal group mice, the DSS model group, and the EHH-ExoNPs intervention group mice. The specific implementation is as follows:

[0080] Extraction of RNA from colon tissue using Trizol method:

[0081] 1) Add 5 mm of colon tissue, five 3 mm grinding beads, and 1 mL of Trizolate to a sterile, enzyme-free grinding tube. Place the tubes symmetrically in a pre-cooled -20°C grinder and grind until no tissue fragments or color remain.

[0082] 2) Transfer the liquid to a new tube (separate from the grinding beads) and place on ice for 5 minutes;

[0083] 3) Add chloroform (1 / 5 of the volume of Trizol), shake vigorously until the mixture turns milkshake color, and let it stand for 10 minutes;

[0084] 4) Centrifuge at 12,000 rpm for 15 minutes at 4°C until stratification is observed. Transfer the upper supernatant to a new EP tube.

[0085] 5) Add pre-cooled isopropanol in an equal volume to the supernatant in step 4 and shake with a shaker;

[0086] 6) After standing for 10 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes;

[0087] 7) Discard the supernatant, remove the sediment from the tube wall by aspirating, and place the tube upside down on a paper towel to dry until the sediment is transparent;

[0088] 8) Add 1 mL of pre-chilled 75% ethanol to wash the DNA, resuspend the precipitate (by flicking with your finger), and centrifuge at 7500 rpm for 5 min at 4°C.

[0089] 9) Same as step 7;

[0090] 10) Dissolve RNA in DEPC water (50 μL) and place on ice for 30 min;

[0091] 11) After RNA is dissolved, mix thoroughly and add 1 μL of the solution to a Nanodrop 2000 spectrophotometer to measure RNA concentration and purity and record the data.

[0092] RNA reverse transcription and real-time quantitative PCR (RT-qPCR): Remove genomic DNA and prepare the reverse transcription system. After thorough mixing, place the cells in a PCR instrument and adjust the program: Step 1: 37°C, 15 minutes; Step 2: 85°C, 5 seconds; 4°C, ∞. After the reaction, the resulting cDNA was premixed with RT-qPCR reagents to prepare the system and amplified in a qPCR instrument. The CT value for each group was obtained. The relative expression level of each target gene was calculated using the 2-ΔΔCT method, using GAPDH as the internal reference.

[0093] From the above, we can see that Figure 10-14qPCR results showed that compared with DSS colitis mice, the relative mRNA expression levels of proinflammatory cytokines IL-6 (EHH-ExoNPs:DSS, P<0.001), TNF-α (EHH-ExoNPs:DSS, P<0.01) and IL-1β (EHH-ExoNPs:DSS, P<0.05) in the intervention group were significantly decreased, and the expression levels of protective barrier-related factors Occludin (EHH-ExoNPs:DSS, P<0.01) and Claudin (EHH-ExoNPs:DSS, P<0.05) were increased in the intervention group.

[0094] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A use of extracellular vesicles derived from Herba Euphorbiae in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The specific steps are as follows: Step 1: Extraction of leaf cells from Euphorbia pulex: Select fresh, healthy Euphorbia pulex leaves as experimental materials and chop them into small pieces. Add the chopped sample to the enzymatic hydrolysis solution, ensuring full contact between the sample and the solution and incubating. Gently flip or stir the sample during incubation to promote the enzymatic hydrolysis process, thereby isolating the leaf cells. The enzymatic hydrolysis temperature for separating leaf cells was set at 45°C, the pH value was 4.5, and the hydrolysis time was 45 minutes. The enzymatic hydrolysis solution was a mixture of 0.6% cellulase and 0.25% pectinase, which degraded the plant cell walls and released the leaf cells. During the enzymatic hydrolysis process, the pH value was maintained stable by using a buffer to protect the leaf cells from damage. Step 2, culturing leaf cells of Herba Euphorbiae Herba: culturing leaf cells of Herba Euphorbiae Herba in a culture medium, so that the leaf cells grow and reproduce normally and secrete extracellular vesicles; The leaf cells of the Herba Euphorbiae are cultured in a culture medium using MS as a basic culture medium under sterile conditions, cytokinin, production factor and 2,4-D growth regulator are added, the pH value of the MS culture medium is adjusted to 5.8-6.0, the culture temperature is 20-28° C., the sucrose concentration is 30 g / L, and during the leaf cell culture period, the light duration is 12 hours, alternating with dark time, and the illumination is 2500 Lx; Step 3, isolation and purification of extracellular vesicles: Leaf cells secrete extracellular vesicles during the culture process. Undigested cell debris and impurities are removed by stepwise low-speed centrifugation at a speed of 300-3500×g. The supernatant after centrifugation is collected, which contains extracellular vesicles. The supernatant is ultracentrifuged at a speed of 100,000×g, the supernatant is discarded, and the extracellular vesicles are resuspended with phosphate buffer. The resuspended extracellular vesicles are added to ultracentrifuge tubes containing 60 wt%, 45 wt%, and 30 wt% sucrose solutions, and then ultracentrifuged at 140,000×g for 60-90 minutes at 4°C. The liquid band between 45 wt% and 30 wt% is aspirated and mixed with PBS. The sucrose solution is removed by further ultracentrifugation at 150,000×g for 60-90 minutes at 4°C. The resulting precipitate is resuspended with PBS to obtain extracellular vesicles derived from Herba Euphorbiae, and then the extracellular vesicles are characterized to determine their size, morphology, and surface marker characteristics. Step 4: Collect and extract the extracellular vesicle fluid and store it in a -20°C refrigerator for future use.

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