Application of extracellular vesicle-like particles derived from divaricate saposhnikovia root in preparation of medicines for preventing and treating intestinal diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of extracellular vesicle-like particles derived from divaricate saposhnikovia roots in preparation of medicines for preventing and treating intestinal diseases. Research on the physiological efficacy of the extracellular vesicle-like particles from divaricate saposhnikovia root shows that the extracellular vesicle-like particles can relieve colon shortening and colon tissue damage caused by dextran sodium sulfate, relieve intestinal inflammation and promote repair of intestinal barriers including mechanical barriers, chemical barriers and immune barriers. The polypeptide has potential for preparing the medicine for treating the intestinal diseases, and a new strategy is provided for preventing or treating the intestinal diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of extracellular vesicle-like particles derived from siler in the preparation of drugs for preventing and treating intestinal diseases. Background Art

[0002] Ulcerative colitis is a chronic, nonspecific inflammatory disease affecting the colon and rectum. Clinical manifestations include persistent or recurrent diarrhea, bloody stools with mucus, abdominal pain, and tenesmus. Severe cases can be complicated by toxic megacolon, intestinal perforation, massive lower gastrointestinal bleeding, and cancer. In recent years, the incidence of ulcerative colitis has shown a significant increase worldwide.

[0003] Intestinal barrier dysfunction is not only the main pathological feature of ulcerative colitis, but also a key target for elucidating the pathogenesis of ulcerative colitis and exploring drugs for the prevention and treatment of ulcerative colitis. The intestinal barrier is a highly selective barrier system present in the intestine, consisting of four parts: mechanical barrier, chemical barrier, biological barrier and immune barrier. An intact intestinal barrier can resist the invasion of pathogens, toxins and antigens. However, when the intestinal barrier is damaged, it can lead to the translocation of bacteria and toxins and immune disorders, resulting in continued increase in intestinal mucosal permeability, triggering and aggravating intestinal inflammation. Currently, the treatment of ulcerative colitis mainly relies on aminosalicylic acid preparations, glucocorticoids, immunosuppressants and biological agents, but there are problems such as poor response, high recurrence rate and significant side effects. Therefore, exploring new treatment strategies for ulcerative colitis has become the focus of current research.

[0004] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by cells that stably carry important biomolecules such as nucleic acids, proteins, and lipids, and participate in the regulation of pathological and physiological processes. Extracellular vesicle-like particles (EVLPs) derived from traditional Chinese medicines have attracted widespread attention due to their wide availability, high yield, good safety, and significant activity. Currently, CHM-EVLPs derived from traditional Chinese medicines include EVs from Houttuynia cordata and EV nanoparticles derived from Atractylodes macrocephala. These CHM-EVLPs have demonstrated therapeutic effects against ulcerative colitis, reducing the expression of inflammatory factors in macrophages and protecting the mucosal barrier. However, intestinal damage caused by ulcerative colitis includes mechanical, chemical, and immune damage, which is multifaceted. Existing CHM-EVLPs can only repair mechanical damage to a limited extent, but cannot achieve comprehensive repair of intestinal damage. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an application of extracellular vesicle-like particles derived from Saposhnikovia divaricata in the preparation of drugs for preventing and treating intestinal diseases. The extracellular vesicle-like particles derived from Saposhnikovia divaricata in the present invention can achieve comprehensive repair of intestinal damage from three aspects: mechanical damage, chemical damage and immune damage, thereby providing a new approach for the treatment of ulcerative colitis.

[0006] The first aspect of the present invention provides an application of extracellular vesicle-like particles derived from Saposhnikovia divaricata in the preparation of drugs for preventing and treating intestinal diseases, wherein the extracellular vesicle-like particles derived from Saposhnikovia divaricata refer to extracellular vesicles extracted from Saposhnikovia divaricata; the drugs for preventing and treating intestinal diseases refer to drugs used for intestinal barrier repair.

[0007] This study investigates the physiological effects of extracellular vesicle-like particles derived from Saposhnikovia divaricata and finds that they can alleviate colon shortening and colon tissue damage caused by sodium dextran sulfate, and promote the repair of intestinal barriers, including mechanical, chemical, and immune barriers. The mechanical barrier is primarily manifested in its ability to promote the expression of tight junction proteins ZO-1, Claudin 1, and Occludin; the chemical barrier is primarily manifested in its ability to promote mucin secretion; and the immune barrier is manifested in its ability to inhibit macrophage M1 polarization and the expression of Th1 cells. Furthermore, the drug exhibits good safety at both the cellular and animal levels.

[0008] In another preferred embodiment, the intestinal disease comprises ulcerative colitis.

[0009] In another preferred embodiment, the intestinal barrier includes intestinal immune barrier, intestinal chemical barrier and physical barrier.

[0010] In another preferred embodiment, the extracellular vesicle-like particles derived from Saposhnikovia divaricata are the only active ingredient in the medicine.

[0011] In another preferred embodiment, the extracellular vesicle-like particles are obtained by the following method: Mix the radix fangfeng with PBS buffer and crush to obtain radix fangfeng slurry; centrifuge the radix fangfeng slurry at 500g-2000g for 20-40min to remove plant fibers and large particles; continue to centrifuge at 3000g-10000g for 60-100min to remove debris and organelles; finally, centrifuge at 100000g-130000g for 60-90min to collect the precipitate; Mix the radix fangfeng with water, grind it to obtain a slurry of radix fangfeng, filter it, and centrifuge the filtrate at 500g-2000g for 20min-40min to remove plant fibers and large particles; continue to centrifuge at 3000g-10000g for 60min-100min to remove debris and organelles; finally, centrifuge at 100000g-130000g for 60min-90min, collect the precipitate, and resuspend the precipitate to obtain a mixed solution; Sucrose was dissolved in water to prepare sucrose solutions with mass percentages ranging from 8% to 60%. The sucrose solutions were added into centrifuge tubes in descending order of mass percentage using a layering method to obtain a sucrose concentration gradient solution. The mixed solution was injected into the sucrose concentration gradient solution and ultracentrifuged. The bands in the sucrose solution with a mass percentage concentration of 30% to 45% were collected to obtain extracellular vesicle-like particles derived from Saposhnikovia divaricata.

[0012] In another preferred embodiment, the mass percentage concentrations of the sucrose concentration gradient solution from bottom to top are 60%, 45%, 30% and 8% respectively.

[0013] The second aspect of the present invention provides an inflammatory factor expression inhibitor, comprising the extracellular vesicle-like particles.

[0014] The third aspect of the present invention provides a drug for preventing ulcerative colitis, comprising the extracellular vesicle-like particles.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This study investigates the physiological effects of extracellular vesicle-like particles derived from fangfeng (Saposhnikovia divaricata). These particles can alleviate dextran sulfate sodium-induced colitis in mice, alleviating colon shortening and tissue damage caused by dextran sulfate sodium. They also promote the repair of intestinal barriers, including mechanical, chemical, and immune. The mechanical barrier is primarily manifested by promoting the expression of tight junction proteins ZO-1, Claudin 1, and Occludin; the chemical barrier is primarily manifested by promoting mucin secretion; and the immune barrier is manifested by inhibiting macrophage M1 polarization and Th1 cell expression. These particles exhibit good safety at both the cellular and animal levels. Experiments further revealed that these particles can scavenge reactive oxygen species within macrophages, inhibit M1 macrophage polarization, and suppress the secretion of proinflammatory cytokines IL-1β, IL-6, and TNF-α. Therefore, these extracellular vesicle-like particles have the potential to be used in the preparation of drugs for treating intestinal diseases, providing a new strategy for the prevention or treatment of intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Transmission electron microscopy images of SR-EVLPs, wherein a is a transmission electron microscopy image of SR-EVLPs, b is an enlarged image of point A in a, and SR-EVLPs are extracellular vesicle-like particles derived from Saposhnikovia divaricata.

[0017] Figure 2 This figure shows the detection results of SR-EVLPs clearing reactive oxygen species in macrophages RAW264.7.

[0018] Figure 3 The figures show the results of different treatment groups on M1 polarization of macrophages RAW264.7; A shows the results of CD80-positive cells; B shows the results of CD86-positive cells.

[0019] Figure 4 Figures A and B show the effects of different treatment groups on the secretion of pro-inflammatory factors by macrophages RAW264.7. Figure A shows the effects of inhibiting the secretion of IL-1β by RAW264.7, Figure B shows the effects of inhibiting the secretion of IL-6 by RAW264.7, and Figure C shows the effects of inhibiting the secretion of TNF-α by RAW264.7.

[0020] Figure 5 Figure 3 is the result of different treatment groups on DSS-induced mouse colon shortening. A is the colon image after treatment in different treatment groups, and B is the quantitative statistical graph of A.

[0021] Figure 6 H&E staining images of the colonic damage of mice induced by DSS in different treatment groups, where the white circles pointed by arrows represent the damaged sites induced by DSS.

[0022] Figure 7 This figure shows the effects of different treatment groups on the expression of tight junction proteins ZO-1, Claudin 1, and Occludin in colon tissue.

[0023] Figure 8 Alcian blue staining of the repair of goblet cells and mucin secretion in colon tissue in different treatment groups.

[0024] Figure 9 The results of the effects of different treatment groups on the polarization of M1 macrophages in the colonic lamina propria.

[0025] Figure 10 This figure shows the effects of different treatment groups on the expression of Th1 cells in mesenteric lymph nodes.

[0026] Figure 11 The toxicity test results of SR-EVLPs with different concentrations on macrophages RAW264.7.

[0027] Figure 12 This is the safety test result of SR-EVLPs on mice. DETAILED DESCRIPTION

[0028] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below.

[0030] Currently, the treatment of ulcerative colitis relies primarily on aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics, but these are associated with poor response, high recurrence rates, and significant side effects. Therefore, exploring new treatment strategies for ulcerative colitis has become a research priority.

[0031] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by cells that stably carry important biomolecules such as nucleic acids, proteins, and lipids, and participate in the regulation of pathological and physiological processes. Extracellular vesicle-like particles (EVLPs) derived from traditional Chinese medicine (TCM) have attracted widespread attention due to their wide availability, high yield, good safety, and significant activity. Currently, EVLPs derived from TCM include those derived from Houttuynia cordata and those derived from Atractylodes macrocephala. Both of these EVLPs can be used to prepare drugs for ulcerative colitis.

[0032] The present invention discovered for the first time through experiments that extracellular vesicle-like particles derived from Saposhnikovia divaricata can alleviate colon shortening and colon tissue damage caused by dextran sodium sulfate (DSS), and promote the repair of intestinal barriers, including mechanical barriers, chemical barriers and immune barriers, providing a new approach for extracellular vesicle-like particles derived from traditional Chinese medicine.

[0033] The radix fangfeng in the following embodiments is the dried root of the umbelliferae plant radix fangfeng, and the drug implementation standard complies with the 2020 edition of the "Chinese Pharmacopoeia".

[0034] Example 1: Preparation of extracellular vesicle-like particles derived from Saposhnikovia divaricata.

[0035] The specific preparation process is as follows: S1. Rinse the dust and impurities on the surface of the windproof with double distilled water, put the cleaned windproof into a wall breaking machine, add PBS buffer with a concentration of 0.01M and a pH value of 7.4 at 4°C, and stir at high speed to obtain windproof slurry.

[0036] S2. Filter the slurry with a strainer and transfer the slurry to a centrifuge tube. Centrifuge at 500g for 10 minutes, 1000g for 10 minutes, and 2000g for 20 minutes at 4°C to eliminate plant fibers and large particles; then centrifuge at 3000g for 30 minutes, 5000g for 30 minutes, and 10000g for 40 minutes to remove debris and organelles; finally, centrifuge at 130000g for 90 minutes to obtain a precipitate.

[0037] S3. Resuspend the precipitate in sterile PBS buffer at 4°C to obtain a mixed solution; mix sucrose with 20mM Tris-HCL to prepare sucrose solutions with mass percentage concentrations of 8wt%, 30wt%, 45wt% and 60wt%, respectively; add the above sucrose solutions into the centrifuge tube from the bottom in ascending order of mass percentage concentration in equal volumes, and then add the mixed solution, perform two ultracentrifugations at 4°C, and each ultracentrifugation is performed at 130,000g for 90 minutes; after ultracentrifugation, take the band between 30wt% and 45wt% to obtain extracellular vesicle-like particles derived from Saposhnikovia divaricata; resuspend the extracellular vesicle-like particles derived from Saposhnikovia divaricata in sterile PBS buffer at 4°C, record them as SR-EVLPs, and store them at -80°C for future use.

[0038] The morphology of SR-EVLPs was characterized by transmission electron microscopy. Figure 1 As shown in the figure, SR-EVLPs showed a typical saucer-like morphology with a particle size of 74 nm.

[0039] Example 2: Effect of SR-EVLPs on the Endogenous Reactive Oxygen Species Levels in RAW264.7 Macrophage Cells The Beyotime Reactive Oxygen Species Detection Kit (fluorescent probe DCFH-DA) was used to assess the endogenous ROS levels in macrophages.

[0040] First, RAW264.7 cells were seeded in 6-well plates and divided into four groups: a negative control group (designated as the Control group), a H2O2 group, a H2O2 + SR-EVLPs (10 ng / mL) group (designated as the SR-EVLPs (10 ng / mL) group, and a H2O2 + SR-EVLPs (100 ng / mL) group (designated as the SR-EVLPs (100 ng / mL) group. The cells were cultured in an incubator for 12 hours to ensure that the cell confluence reached 50% to 70% at the time of testing.

[0041] The H2O2 concentration was 600 μmol / L in the H2O2, SR-EVLPs (10 ng / mL), and SR-EVLPs (100 ng / mL) groups. The final concentrations of SR-EVLPs in the SR-EVLPs (10 ng / mL) and SR-EVLPs (100 ng / mL) groups were 10 ng / mL and 100 ng / mL, respectively. The cells were incubated in a 37°C cell culture incubator for 4 hours.

[0042] Dilute DCFH-DA with serum-free culture medium to a final concentration of 10 μmol / L to obtain DCFH-DA working solution. Aspirate the cell culture medium and add the diluted DCFH-DA working solution. The volume added should be sufficient to cover the cells. Add at least 1 mL of DCFH-DA working solution to each well of the 6-well plate and incubate in a 37°C cell culture incubator in the dark for 30 minutes. Wash the cells three times with PBS buffer to fully remove the DCFH-DA that has not entered the cells. Collect the cells for flow cytometry detection. The results are as follows Figure 2 As shown in the figure, compared with the Control group, the number of ROS-positive cells in the H2O2 group increased significantly, while the number of ROS-positive cells decreased significantly after administration of SR-EVLPs, indicating that SR-EVLPs can reduce the increase of intracellular reactive oxygen species caused by H2O2.

[0043] Example 3: Effect of SR-EVLPs on M1 polarization of macrophage RAW264.7 cells.

[0044] First, take 2×10 5 RAW264.7 cells were seeded into 12-well plates at a density of 1 μg / well. The experiment was divided into 4 groups, namely the negative control group, the lipopolysaccharide group, the LPS group, the LPS+SR-EVLPs (10 ng / mL) group, and the LPS+SR-EVLPs (100 ng / mL) group. The 12-well plates were placed in a 37°C cell culture incubator for 24 hours. Among them, the LPS concentration in the LPS group, the SR-EVLPs (10 ng / mL) group, and the SR-EVLPs (100 ng / mL) group was 1 μg / mL; in the SR-EVLPs (10 ng / mL) group and the SR-EVLPs (100 ng / mL) group, the SR-EVLPs concentration was 10 ng / mL and 100 ng / mL, respectively, and they were placed in the incubator and incubated for 12 hours.

[0045] The culture medium in the 12-well plate was removed and washed three times with PBS. The cells were collected and centrifuged at 1000g for 5 minutes. The supernatant was discarded and the cells were resuspended in 200μL of PBS. 1μL of CD80 / CD86 flow cytometry antibody was added to each tube and incubated in a dark place at 4℃ for 30 minutes. After incubation, the cells were washed three times with 4℃ PBS and collected for flow cytometry. The results are shown in Figure 2. Figure 3 As shown in the figure, compared with the control group, the number of CD80 and CD86 positive cells in the LPS group increased significantly, while the number of CD80 and CD86 positive cells in the SR-EVLPs (10 ng / mL) and SR-EVLPs (100 ng / mL) groups administered with SR-EVLPs decreased significantly. CD80 and CD86 are commonly used surface markers of M1 macrophages, which indicates that SR-EVLPs can inhibit LPS-induced macrophage M1 polarization.

[0046] Example 4: Effect of SR-EVLPs on the production of proinflammatory cytokines in macrophages RAW264.7.

[0047] First, take 5×10 5 RAW264.7 cells were seeded in a 6-well plate at a density of 100 cells / well. The experiment was divided into 4 groups, namely, Control group, LPS group, LPS + SR-EVLPs (10 ng / mL) recorded as SR-EVLPs (10 ng / mL) group and LPS + SR-EVLPs (100 ng / mL) recorded as SR-EVLPs (100 ng / mL) group. The 6-well plate was placed in a cell culture incubator at 37 ° C for 24 hours.

[0048] Among them, the concentration of LPS in the LPS group, SR-EVLPs (10 ng / mL) group, and SR-EVLPs (100 ng / mL) group was 1 μg / mL; the final concentration of SR-EVLPs in the SR-EVLPs (10 ng / mL) group and SR-EVLPs (100 ng / mL) group was 10 ng / mL and 100 ng / mL, respectively. The cells were incubated in an incubator for 12 hours. After the incubation, RNA extraction kit (Biyuntian, spin column RNAeasy TMRNA was extracted using an animal RNA extraction kit. First, remove the medium from the 6-well plate and wash three times with PBS. Collect the cells and centrifuge at 1000g for 2 minutes. Discard the supernatant. Add 300μL of lysis buffer to each tube and gently pipette to transfer the solution to a clean centrifuge tube. Add an equal volume of binding buffer to the lysate and gently invert five times to mix thoroughly. Transfer the mixture to a purification column and centrifuge at 12000g for 30 seconds. Discard the liquid in the collection tube. Add 600μL of wash buffer I and centrifuge at 12000g for 30 seconds. Discard the liquid in the collection tube. Add 600μL of wash buffer II and centrifuge at 12000g for 30 seconds. Discard the liquid in the collection tube. Add 600μL of wash buffer II and centrifuge at 12000g for 30 seconds. Discard the liquid in the collection tube. Centrifuge at 15000g for 2 minutes to remove any residual liquid. Place the RNA purification column in the RNA elution tube, add 40 μL of elution buffer, leave at room temperature for 3 minutes, and centrifuge at 15,000 g for 30 seconds. The resulting solution is the purified RNA.

[0049] Set up the reverse transcription system, add 1 μg of RNA, 4 μL of 5× cDNA first-strand synthesis premix, make up the system to 20 μL with dimethylnitrosoacetamide water, and perform the reverse transcription reaction on a PCR instrument according to the following reaction program: 15 minutes at 55°C, 5 minutes at 85°C, and maintain at 4°C.

[0050] After the reverse transcription is completed, add 180 μL of dimethylnitrosoacetamide water to the above system, mix the above mixture with a pipette, and transfer 15 μL of PCR mixture to the 8-row PCR reaction tube. Add 5 μL of DNA template (GADPH / IL-1β / IL-6 / TNF-α) to each tube and mix carefully with a pipette. Place the reaction tank on the fluorescent quantitative PCR instrument, set and start according to the following program. Pre-denaturation: 95°C, 10 minutes; denaturation: 95°C, 15 seconds; annealing: 60°C, 30 seconds; extension: 72°C, 30 seconds, 40 cycles, and finally keep at 4°C. The test results are as follows: Figure 4 As shown in the figure, compared with the control group, the mRNA expression levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α in the LPS group were significantly increased, while the mRNA expression levels of these proinflammatory cytokines in the SR-EVLPs (10 ng / mL) group and the SR-EVLPs (100 ng / mL) group were significantly decreased. This indicates that SR-EVLPs can inhibit the expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α induced by LPS.

[0051] Example 5: Construction of DSS-induced chronic colitis model in mice.

[0052] Eight-week-old C57BL / 6 male mice were selected and adapted for one week. The mice were randomly divided into four groups: control group, DSS group, DSS+SR-EVLPs (5 mg / kg) group, and DSS+SR-EVLPs (10 mg / kg) group. Among them, the DSS group drank 2.5wt% DSS solution for 7 days, and the DSS solution was replaced with purified water on the 8th day for 7 days; the DSS+SR-EVLPs (5 mg / kg) group drank the same as the DSS group for the first 7 days, and on the 8th day, the 2.5wt% DSS solution was replaced with SR-EVLPs at a dose of 5 mg / kg for 7 days; the DSS+SR-EVLPs (10 mg / kg) group drank the same as the DSS group for the first 7 days, and on the 8th day, the 2.5wt% DSS solution was replaced with SR-EVLPs at a dose of 10 mg / kg for 7 days; the previous steps were repeated three times. On the last day of the last cycle, the mice were killed and the colon tissues were dissected. The colon length of each group of mice was measured and statistically analyzed. Figure 5 As shown in the figure, compared with the control group, the colon length of mice in the DSS group was significantly shortened, while the colon length of mice in the DSS+SR-EVLPs (5 mg / kg) group and the DSS+SR-EVLPs (10 mg / kg) group given SR-EVLPs increased significantly after oral treatment. This indicates that SR-EVLPs can improve the colon shortening caused by DSS.

[0053] Example 6: Effects of SR-EVLPs on colon tissue pathology.

[0054] The colon tissue obtained after dissection was fixed and then immersed in 70wt% ethanol solution, 80wt% ethanol solution and 85wt% ethanol solution for 30 minutes each, 95wt% ethanol solution for 40 minutes, anhydrous ethanol for 40 minutes three times, xylene for 25 minutes, xylene at 45°C for 30 minutes, wax tanks I and II at 65°C for 30 minutes each, and wax tank III for 40 minutes for dehydration. The tissue was then embedded at 60°C.

[0055] Use a microtome to slice the wax block into sections continuously, place the wax strips in warm water to stretch them, make them adhere to the slides and bake them to dry.

[0056] The sections were sequentially placed in xylene I and xylene II for 10 minutes each, anhydrous ethanol for 5 minutes, 95% ethanol solution, 85% ethanol solution and 75% ethanol solution for 3 minutes each, washed with water, hematoxylin for 4 minutes, washed with water, 1% acidic ethanol differentiation solution for 30 seconds, washed with water, ammonia for 10 seconds, washed with water, eosin staining solution for 4 minutes → washed with water, 95% ethanol solution I and II for 3 minutes each, anhydrous ethanol 2 times for 3 minutes each, xylene 3 times for 3 minutes each. After the sections were dried, they were sealed with neutral gum and observed and photographed under a microscope. The results are as follows Figure 6 As shown in the figure, compared with the control group, the mice in the DSS group showed destroyed colon tissue structure, crypt distortion, reduced crypt number, and inflammatory cell infiltration, while these symptoms were significantly alleviated after oral treatment with SR-EVLPs. This indicates that SR-EVLPs can improve the colon pathological damage caused by DSS.

[0057] Example 7: Effects of SR-EVLPs on the intestinal physical barrier.

[0058] Immunofluorescence staining was performed using frozen sections of colon tissue. First, the samples were fixed with methanol at -20°C for 10 minutes at room temperature. Then, the samples were washed 3 times with 1×PBS for 5 minutes each time. The colon tissues in the Control group, DSS group, DSS+SR-EVLPs (5 mg / kg) group, and DSS+SR-EVLPs (10 mg / kg) group were placed in 0.2% Triton X-100 and incubated at room temperature for 5 minutes. The samples were washed again with 1×PBS 3 times for 5 minutes each time. Use an immunohistochemistry pen to draw a circle around the sample, add 5% goat serum to the surface of the sample and block at room temperature for 1 hour. After blocking, wash off the blocking solution, add the fluorescent primary antibody of ZO-1 / Claudin 1 / Occludin to the surface of the sample, and incubate overnight at 4°C. Finally, wash the sample with 1×PBS, add an anti-fluorescence quencher containing DAPI to the surface of the sample and seal the slide, and observe and photograph it under a fluorescence microscope. The results are as follows Figure 7 As shown in the figure, compared with the control group, the expression of tight junction proteins ZO-1 / Claudin1 / Occludin in the colon tissue of mice in the DSS group was significantly reduced; however, after oral treatment with SR-EVLPs, the expression of these tight junction proteins was significantly increased. This indicates that SR-EVLPs can promote the expression of tight junction proteins ZO-1 / Claudin 1 / Occludin and promote the repair of the intestinal mechanical barrier.

[0059] Example 8: Effects of SR-EVLPs on the intestinal chemical barrier.

[0060] Alcian blue staining kit was used to stain the paraffin sections of colon tissue in the Control group, DSS group, DSS+SR-EVLPs (5 mg / kg) group and DSS+SR-EVLPs (10 mg / kg) group with Alcian blue. First, the sections were dewaxed to water, then Alcian acidification solution was added to soak the sample for 3 minutes, rinsed with running water, and then Alcian staining solution was added for 30 minutes. After rinsing with running water again, nuclear fast red staining solution was added for counterstaining for 5 minutes, rinsed with running water for 1 minute, and then placed in gradient ethanol for dehydration. Xylene was used for transparency, and finally the sections were sealed with neutral gum and observed under a microscope for pictures. Mucin was blue and the cell nucleus was red. The results are as follows Figure 8 As shown in the figure, compared with the control group, the goblet cells in the colon tissue of mice in the DSS group were significantly reduced and damaged, and mucin secretion was reduced; however, after oral treatment with SR-EVLPs, the number of goblet cells increased and mucin secretion decreased. This suggests that SR-EVLPs can promote the repair of goblet cell damage and mucin secretion, promoting the repair of the intestinal chemical barrier.

[0061] Example 9: Effect of SR-EVLPs on M1 polarization of colonic lamina propria macrophages.

[0062] Remove the mouse colon, cut the intestinal lumen longitudinally with scissors, and flush the intestinal contents. Prepare a 1mM DTT solution (Solution 1) and a 30mM colon EDTA solution (Solution 2) in PBS. Place the colon tissue in a centrifuge tube containing Solution 1 and shake vigorously on a horizontal shaker at 220 rpm and 37°C for 10 minutes. Remove the colon tissue and wash it with PBS. After washing, transfer the colon to a centrifuge tube containing Solution 2 and shake vigorously on a horizontal shaker at 220 rpm and 37°C for 10 minutes.

[0063] Remove the centrifuge tube from the horizontal shaker, remove the colon tissue, and transfer it to 1640 complete medium for washing. Prepare a digestion solution containing 150 μg / mL DNase I and 200 U / mL collagenase VIII in 1640 medium. Place the colon tissue in a 6-well plate containing the digestion solution, mince, and digest in an incubator for 90 minutes.

[0064] After digestion, filter the tissue through a 70 μm filter, collect the filtrate, and centrifuge at 500 g for 5 minutes at room temperature to obtain a cell pellet. Resuspend the cell pellet in 40% Percoll and transfer it to a 15 mL centrifuge tube. 80 wt% Percoll is pipetted onto the bottom of the tube and slowly added to form a density gradient. Centrifuge at 800 g for 20 minutes at 4°C. After centrifugation, aspirate the intermediate layer of lymphocytes, centrifuge, and remove the supernatant. The resulting cell pellet is the intestinal lamina propria lymphocytes. Resuspend the cells in Flowing Buffer, transfer them to a flow cytometry tube, centrifuge, and remove the supernatant.

[0065] CD45, CD11b, F4 / 80 and CD86 flow cytometry antibodies were used to label colonic M1 macrophages in the intestinal lamina propria lymphocytes. The antibodies were diluted with Flowing Buffer at a volume ratio of 1:200. 200 μL of the diluted antibodies were added to each tube and incubated in the dark at 4°C for 30 minutes. After incubation, the residual antibodies were washed with Flowing Buffer, the supernatant was removed by centrifugation, and the cell pellet was resuspended with Flowing Buffer and detected by flow cytometry. The results are shown in Figure 2. Figure 9 As shown in the figure, cells that are double positive for F4 / 80 and CD86 are M1 macrophages. Compared with the control group, the number of M1 macrophages in the colon tissue of mice in the DSS group increased significantly. However, after oral treatment with SR-EVLPs, the number of M1 macrophages decreased significantly. This indicates that SR-EVLPs can inhibit DSS-induced M1 polarization of colonic lamina propria macrophages and promote the repair of the intestinal immune barrier.

[0066] Example 10: Effect of SR-EVLPs on Th1 cells in mesenteric lymph nodes.

[0067] Lymph nodes from the mouse mesenteric membrane were collected and ground through a 70 μm filter. The filter was rinsed with PBS to collect lymphocytes. The cells were centrifuged at 600 g for 5 minutes at 4°C and the supernatant was discarded. The resulting pellet was the mesenteric lymphocytes.

[0068] Resuspend the cells in PBS and count the viable cells. Adjust the cell density to 1 × 10 cells / mL using 1640 complete medium. 6 Cells were seeded in 96-well plates at 2×10 cells / mL. 5 cells. Join eBioscience TM Add the cell stimulation mixture and incubate in a cell culture incubator for 4-6 hours. After incubation, collect the cells and centrifuge to discard the supernatant.

[0069] Use CD45, CD4 and IFN-γ antibodies to label colon Th1 cells in mesenteric lymph nodes. Dilute the antibodies at a ratio of antibody: Flowing Buffer = 1:200. Use CD45 and CD4 to perform surface antibody staining first, add 200μL of diluted antibody to each tube, incubate at 4°C in the dark for 30 minutes, wash the residual antibody with Flowing Buffer after the incubation, add 250μL of Fixation Buffer to each tube, incubate at 4°C in the dark for 30 minutes. After the incubation, wash with Washing Buffer, centrifuge and remove the supernatant, add 200μL of diluted intracellular antibody IFN-γ to each tube, incubate at 4°C in the dark for 30 minutes, finally wash with Flowing Buffer and resuspend the cell pellet, and detect using flow cytometry. The results are as follows. Figure 10 As shown in the figure, cells that are double positive for CD4 and INF-γ are Th1 cells. Compared with the control group, the number of Th1 cells in the mesenteric lymph nodes of mice in the DSS group increased significantly. However, after oral treatment with SR-EVLPs, the number of Th1 cells decreased significantly. This indicates that SR-EVLPs can inhibit the increase of Th1 cells in the mesenteric lymph nodes induced by DSS and promote the repair of the intestinal immune barrier.

[0070] Example 11: Effect of SR-EVLPs on macrophage cell viability.

[0071] Cell viability was assessed using the CCK8 assay. RAW264.7 macrophages were seeded at 3,000 cells / well in a 96-well plate. The experiment was divided into six groups, one of which served as a negative control group. The other five groups were treated with 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL of SR-EVLPs, respectively. The volume of the cell suspension in each well was 200 μL.

[0072] Place the 96-well plate in a 37°C incubator for 24 hours, then remove and remove the culture medium. Prepare a culture medium containing CCK-8 (CCK-8: culture medium = 1:10), and add 100 μL of culture medium containing CCK-8 to each well along the well wall. Be careful not to produce bubbles in the well to avoid affecting the test results. Place in a 37°C incubator for 1 hour, and measure the absorbance value at 450 nm using a microplate reader. The results are as follows: Figure 11 As shown in the results, 10-200 μg / mL SR-EVLPs had no effect on the cell activity of macrophage RAW264.7, indicating that SR-EVLPs has good safety at the cellular level.

[0073] Example 12: Effects of SR-EVLPs on the heart, liver, spleen, lungs, and kidneys of mice.

[0074] Eight-week-old C57BL / 6 male mice were selected and adaptively raised for one week. The mice were randomly divided into three groups, namely the Control group, the 5mg / kg SR-EVLPs group and the 10mg / kg SR-EVLPs group. The mice in the 5mg / kg SR-EVLPs group and the 10mg / kg SR-EVLPs group were gavaged with SR-EVLPs every other day, at doses of 5mg per kilogram of body weight and 10mg per kilogram of body weight, respectively, for 30 consecutive days. On the last day, the mice were killed, and the heart, liver, spleen, lungs, and kidneys were dissected and fixed, embedded, sliced, and HE-stained according to the method of Example 6. They were observed under a microscope and photographed. The results are shown in Figure 6. Figure 12 As shown in the results, oral administration of 5 mg and 10 mg of SR-EVLPs per kg body weight to mice did not cause significant damage to the heart, liver, spleen, lungs, or kidneys. This indicates that SR-EVLPs has good safety at the animal level.

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A use of extracellular vesicle-like particles derived from Saposhnikovia divaricata in the preparation of a drug for preventing and treating intestinal diseases, characterized in that: The extracellular vesicle-like particles derived from fangfeng refer to extracellular vesicles extracted from fangfeng; The drug for preventing and treating intestinal diseases refers to a drug used for repairing the intestinal barrier.

2. The use according to claim 1, characterized in that The intestinal diseases include ulcerative colitis.

3. The use according to claim 1, characterized in that The intestinal barrier includes intestinal immune barrier, intestinal chemical barrier and intestinal physical barrier.

4. The use according to claim 1, characterized in that The medicine contains extracellular vesicle-like particles derived from fangfeng as the only active ingredient.

5. The use according to any one of claims 1 to 4, characterized in that The extracellular vesicle-like particles derived from the siler are obtained by the following method: The radix fangfeng is mixed with PBS buffer and crushed to obtain a slurry of radix fangfeng; the slurry of radix fangfeng is centrifuged at 500g-2000g for 20min-40min to remove plant fibers and large particles; the slurry is further centrifuged at 3000g-10000g for 60min-100min to remove debris and organelles; and finally, the precipitate is collected and resuspended to obtain a mixed solution by centrifugation at 100000g-130000g for 60min-90min. Sucrose was dissolved in water to prepare sucrose solutions with mass percentages ranging from 8% to 60%. The sucrose solutions were added into centrifuge tubes in descending order of mass percentage using a layering method to obtain a sucrose concentration gradient solution. The mixed solution was injected into the sucrose concentration gradient solution and ultracentrifuged. The bands in the sucrose solution with a mass percentage concentration of 30% to 45% were collected to obtain extracellular vesicle-like particles derived from Saposhnikovia divaricata.

6. The use according to claim 5, characterized in that The ultracentrifugation conditions are 100,000 g to 130,000 g for 60 min to 90 min.

7. The use according to claim 5, characterized in that The mass percentage concentrations of the sucrose concentration gradient solution from bottom to top are 60%, 45%, 30% and 8% in sequence.

8. An inflammatory factor expression inhibitor, characterized in that The invention comprises the extracellular vesicle-like particles described in claim 1.

9. A drug for preventing ulcerative colitis, characterized in that: The invention comprises the extracellular vesicle-like particles described in claim 1.

Images