Application of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of drugs for preventing or treating intestinal diseases

Abstract

The present invention relates to the use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of drugs for preventing or treating intestinal diseases. The present invention creatively extracts and purifies extracellular vesicles derived from the traditional Chinese medicine Atractylodes macrocephala, and studies its physiological effects, and finds that it can be fully internalized and absorbed by macrophages, can reduce the expression level of inflammatory factors in macrophages, and has in vivo intestinal targeting. Animal experiments have proved that it can reduce the disease activity index of ulcerative colitis mouse models, alleviate the destruction and atrophy of crypt glands in colon tissue, and reduce the expression level of inflammatory factors in mouse colon tissue. It has the potential to be used in the preparation of drugs for preventing or treating intestinal diseases, and provides a new strategy for preventing or treating intestinal diseases, especially enteritis, ulcerative enteritis, and ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a new drug strategy for preventing or treating intestinal diseases, and specifically to the use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of drugs for preventing or treating intestinal diseases. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease characterized by diffuse lesions of the colonic mucosa. Currently, the commonly used therapeutic drugs in Western medicine are aminosalicylate, corticosteroids and immunosuppressants, which are of various types but have great side effects, are prone to recurrence, and cause adverse conditions such as drug dependence. Atractylodes macrocephala, first recorded in Shennong's Herbal Classic, Volume II, Top Grade, is bitter, warm, and belongs to the spleen and stomach meridians. It can replenish qi and strengthen the spleen, dry dampness and promote diuresis, stop sweating and stabilize pregnancy. It has a wide range of clinical applications and high pharmaceutical value. Atractylodes macrocephala is now being developed for clinical applications in a variety of diseases because of its medicinal and edible nature, low price and rich pharmacological properties.

[0003] Vesicle-like nanoparticles (VLNs) contain a variety of proteins, lipids, and nucleic acids, and can play important physiological functions by mediating cell-to-cell communication. Almost all types of eukaryotic and prokaryotic cells secrete VLNs. Plant VLNs are similar to mammalian VLNs in morphology, but their composition and function have been less studied. Studies have found that plant VLNs are components of the plant innate immune system and have antifungal effects. In addition, plant VLNs have cross-species regulatory functions, which can not only regulate the physiological functions of mammalian cells, but also intervene and prevent disease progression and play a therapeutic role in diseases. These research conclusions prove that plant VLNs, as a new type of natural product, may become a good candidate source for new drug development.

[0004] Traditional Chinese medicine is mostly plant-based, low-cost, and has few side effects, but few people have studied EVs derived from traditional Chinese medicine. It is still unknown whether it is feasible to extract extracellular vesicles from the traditional Chinese medicine Atractylodes macrocephala, and whether the extracted Atractylodes macrocephala-derived extracellular vesicle-like nanoparticles have biological activity. Therefore, it is meaningful to provide a method for extracting high-purity, high-quality extracellular vesicles from the traditional Chinese medicine Atractylodes macrocephala, and to conduct in-depth research on the functions of the extracted extracellular vesicle-like nanoparticles to explore their application potential in the treatment of diseases. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a new drug strategy for preventing or treating intestinal diseases, specifically to provide the use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of drugs for preventing or treating intestinal diseases.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of drugs for preventing or treating intestinal diseases.

[0008] The present invention creatively extracts and purifies the extracellular vesicles derived from the Chinese herbal medicine Atractylodes macrocephala, and studies its physiological effects, and finds that it can be fully internalized and absorbed by macrophages, can reduce the expression levels of inflammatory factors such as IL-1β, IL-6 and COX-2 in macrophages, exerts anti-inflammatory and antioxidant effects, and has in vivo intestinal targeting. Animal experiments have shown that it can reduce the disease activity index of ulcerative colitis (UC) mouse models, alleviate the destruction and atrophy of crypt glands in colon tissue, and reduce the expression level of inflammatory factors in mouse colon tissue. It has the potential to be used in the preparation of drugs for the prevention or treatment of intestinal diseases, and provides a new strategy for the prevention or treatment of intestinal diseases, especially enteritis, ulcerative enteritis, and ulcerative colitis.

[0009] Preferably, the type of intestinal disease includes enteritis, preferably ulcerative enteritis, and more preferably ulcerative colitis.

[0010] The extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala involved in the present invention can be prepared by any method known to those skilled in the art, and the following extraction method is more preferred. The extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala obtained by this method have higher purity and better effect in preventing and treating intestinal diseases.

[0011] Preferably, the extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala are prepared by an extraction method comprising the following steps:

[0012] (1) filtering the juice of the Atractylodes macrocephala raw material, centrifuging the filtrate and collecting the supernatant;

[0013] (2) Filter the supernatant, perform ultracentrifugation, collect the precipitate and resuspend it to obtain the product.

[0014] Preferably, the centrifugation and collection of the supernatant in step (1) comprises at least four times, and the centrifugation speed is 300-800g (e.g., 300g, 350g, 400g, 450g, 500g, 600g, 700g, 800g, etc.), 1000-4000g (e.g., 1000g, 1200g, 1500g, 1700g, 2000g, 2500g, 3000g, 3500g, 4000g, etc.), 5000-8000g (e.g., 5000g, 5500g, etc.) , 6000g, 6500g, 7000g, 7500g, 8000g, etc.), 9000-15000g (for example, 9000g, 9500g, 10000g, 10500g, 11000g, 11500g, 12000g, 15000g, etc.), and the time of each centrifugation is independently selected from 5-70min (for example, 5min, 10min, 15min, 20min, 30min, 40min, 50min, 60min, 70min, etc.).

[0015] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0016] Further preferably, the centrifugation and collection of the supernatant in step (1) are performed four times in total, the speed of the first centrifugation is 400-600g (e.g., 400g, 450g, 500g, 550g, 600g, etc.), and the time is 5-15min (e.g., 5min, 8min, 10min, 12min, 15min, etc.); the speed of the second centrifugation is 1000-3000g (e.g., 1000g, 1500g, 2000g, 2500g, 3000g, etc.), and the time is 15-25min (e.g., 15min, 18min, 20min, 22min, etc.). in, 25min, etc.); the speed of the third centrifugation is 5000-7000g (for example, 5000g, 5500g, 6000g, 6500g, 7000g, etc.), and the time is 20-40min (for example, 20min, 25min, 30min, 35min, 40min, etc.); the speed of the fourth centrifugation is 9000-12000g (for example, 9000g, 10000g, 11000g, 12000g, etc.), and the time is 50-70min (for example, 50min, 55min, 60min, 65min, 70min, etc.).

[0017] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0018] Preferably, the ultracentrifugation speed in step (2) is 90000-150000 g (e.g., 90000 g, 100000 g, 110000 g, 120000 g, 130000 g, 140000 g, 150000 g, etc.), and the time is 50-100 min (e.g., 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, 100 min, etc.).

[0019] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0020] Preferably, the resuspension is followed by filtering using a 0.22 μm filter.

[0021] In the present invention, the dosage form of the drug includes any one of dispersible tablets, sustained-release tablets, effervescent tablets, enteric-coated tablets, sustained-release capsules, enteric-coated capsules, granules, dripping pills or solutions.

[0022] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0023] 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.

[0024] In a second aspect, the present invention provides the use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of intestinal targeted preparations.

[0025] The present invention also creatively discovered that the extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala have intestinal targeting in vivo. This discovery can be further applied to the preparation of intestinal targeted preparations to study the pathogenesis of intestinal diseases and treat related diseases.

[0026] Preferably, the intestinal targeted preparation further comprises other drugs for preventing or treating intestinal diseases loaded in the extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala.

[0027] In a third aspect, the present invention also provides the use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of inhibitors of inflammatory factor expression.

[0028] In a fourth aspect, the present invention also provides the use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala for non-therapeutic purposes in the preparation of inhibitors of inflammatory factor expression.

[0029] According to the research results of the present invention, the extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala can reduce the expression of inflammatory factors at the cellular level (in vitro level), that is, the extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala can be made into a simple experimental preparation for exploring the expression behavior of macrophage inflammatory factors, the mechanism of changes in inflammatory factors in intestinal tissue and other related fields. The inflammatory factor expression 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 factor expression inhibitors for non-therapeutic purposes.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention creatively extracts and purifies the extracellular vesicles derived from the Chinese herbal medicine Atractylodes macrocephala, and studies its physiological effects, and finds that it can be fully internalized and absorbed by macrophages, can reduce the expression levels of inflammatory factors such as IL-1β, IL-6 and COX-2 in macrophages, exerts anti-inflammatory and antioxidant effects, and has in vivo intestinal targeting. Animal experiments have shown that it can reduce the disease activity index of ulcerative colitis (UC) mouse models, alleviate the destruction and atrophy of crypt glands in colon tissue, and reduce the expression level of inflammatory factors in mouse colon tissue. It has the potential to be used in the preparation of drugs for the prevention or treatment of intestinal diseases, and provides a new strategy for the prevention or treatment of intestinal diseases, especially enteritis, ulcerative enteritis, and ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a transmission electron micrograph of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs);

[0033] Figure 2 is the size distribution of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs);

[0034] Figure 3 This is a graph showing the purity test results of extracellular vesicles (AMVLNs) derived from Atractylodes macrocephala;

[0035] Figure 4 This is a diagram of the detection results of nucleic acids in extracellular vesicles (AMVLNs) derived from Atractylodes macrocephala;

[0036] Figure 5 This is a graph showing the results of lipid detection in extracellular vesicles (AMVLNs) derived from Atractylodes macrocephala;

[0037] Figure 6 This is the result of fluorescence microscopy detection of AMVLNs being internalized and absorbed by RAW264.7;

[0038] Figure 7This is the result of cell flow cytometry detection of AMVLNs internalized and absorbed by RAW264.7;

[0039] Figure 8 This is the result diagram of the effect of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs) intervention on the expression level of inflammatory factors in RAW264.7;

[0040] Fig. 9 This is the result of flow cytometry detection of the effect of AMVLNs on the anti-inflammatory effect of RAW264.7;

[0041] Fig.10 This is the result of flow cytometry detection of the effect of AMVLNs on the antioxidant effect of RAW264.7;

[0042] Fig.11 This is a diagram showing the distribution of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs) in the heart, liver, spleen, lung, kidney, femur, and colon of mice;

[0043] Fig.12 is the anatomical diagram of the colon tissue of each group of mice in the in vivo treatment experiment;

[0044] Fig.13 This is a statistical result diagram of the colon length of each group of mice in the in vivo treatment experiment;

[0045] Fig.14 This is a statistical result diagram of the body weight of each group of mice in the in vivo treatment experiment;

[0046] Fig.15 It is a statistical result diagram of the disease activity index of each group of mice in the in vivo treatment experiment;

[0047] Fig.16 This is the HE staining result of colon tissue of each group of mice in the in vivo treatment experiment;

[0048] Fig.17 is the anatomical diagram of the colon tissue of each group of mice in the in vivo prevention test;

[0049] Fig.18 This is a statistical result diagram of the colon length of each group of mice in the in vivo prevention test;

[0050] Fig.19 This is a statistical result diagram of the body weight of each group of mice in the in vivo prevention test;

[0051] Fig. 20 This is a statistical result graph of the disease activity index of each group of mice in the in vivo prevention test;

[0052] Fig.21 This is the HE staining result of colon tissue of each group of mice in the in vivo prevention test;

[0053] Fig. 22 This is a graph showing the expression levels of inflammatory factors in the colon tissue of each group of mice in the in vivo prevention experiment. DETAILED DESCRIPTION

[0054] 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.

[0055] The raw materials of Atractylodes macrocephala involved in the following examples were purchased from Zhixin Pharmaceutical Co., Ltd., and were originally produced in Zhejiang. They were identified as the dried rhizomes of Atractylodes macrocephala Koidz. by the Chinese Medicine Pharmacy of the Third Affiliated Hospital of Guangzhou University of Chinese Medicine.

[0056] Example 1

[0057] Extraction of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs):

[0058] (1) Take 50 g of Atractylodes macrocephala and squeeze the juice. After filtering, collect the supernatant in a clean 50 mL centrifuge tube and sterilize it;

[0059] (2) Centrifuge at 500 g for 10 min at 4°C; collect the supernatant in a 50 mL centrifuge tube and centrifuge at 2000 g for 20 min at 4°C; collect the supernatant in a 50 mL centrifuge tube and centrifuge at 5000 g for 30 min at 4°C; collect the supernatant in a 50 mL centrifuge tube and centrifuge at 10000 g for 60 min at 4°C;

[0060] (3) The supernatant was filtered and collected in a special centrifuge tube of an ultra-high speed centrifuge. The supernatant was centrifuged at 100,000 g for 70 min at 4°C. The supernatant was discarded, and the precipitate was resuspended in pre-cooled 1× PBS buffer and filtered using a 0.22 μm disposable syringe filter. AMVLNs were collected in a sterilized EP tube and stored in a -80°C refrigerator for later use.

[0061] Example 2

[0062] Characterization of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs):

[0063] The morphology, particle size, purity and chemical composition of the AMVLNs extracted in Example 1 were characterized.

[0064] (1) The morphology of AMVLNs was observed under a transmission electron microscope. Figure 1 As shown: the surface is concave, showing a typical cup-shaped and disc-shaped vesicle structure, the cell membrane structure is clearly visible, the membrane boundary is clear and sharp, the staining background is clean, and the contrast is obvious.

[0065] (2) The particle size distribution of AMVLNs was analyzed using a nanoparticle tracer analyzer. Figure 2 As shown: the particle size is 77.23±18.08nm, between 0-300nm, which is consistent with the extracellular vesicle particle size range reported in the literature, and the concentration is 6.27×10 11 Particles / mL.

[0066] (3) Using Triton X-100 to disrupt the membrane to detect the purity of AMVLNs: After using different concentrations of Triton X-100 to dissolve the membrane of AMVLNs, the change in the number of particles was tested using a nanoflow cytometer. The results are as follows: Figure 3 As shown, the results showed that the purity of the extracted AMVLNs was as high as 78%.

[0067] (4) Agarose gel electrophoresis and thin layer chromatography were used to detect DNA, RNA and lipids in AMVLNs. Figure 4 and Figure 5 As shown (where Figure 4 For nucleic acid results, Figure 5 The results are for lipids), showing that AMVLNs are rich in nucleic acids and lipids.

[0068] The above results prove that the AMVLNs we extracted not only have typical EVs morphology, particle size, and chemical composition, but also have the characteristics of high purity.

[0069] Example 3

[0070] Internalization and absorption test of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs):

[0071] The full internalization and absorption of AMVLNs by RAW264.7 is the prerequisite for the subsequent action of AMVLNs on RAW264.7. Therefore, before doing subsequent experiments, we first verified whether AMVLNs can be internalized and absorbed by RAW264.7. The RAW264.7 cells used in the experiment were donated by the Laboratory of the Department of Laboratory Medicine, Southern Medical University.

[0072] (1) The protein content of AMVLNs was detected by BCA kit: the protein concentration was 1700 μg / mL. In the subsequent research, we used protein concentration to measure the amount of AMVLNs used.

[0073] (2) Preparation of Dil-AMVLNs solution: Take an appropriate amount of AMVLNs and add 1×PBS buffer to 1 mL. After mixing, add 10 μL DiI dye and label at 37°C in the dark for 30 min. Centrifuge at 4°C, 135,000×g for 70 min, discard the supernatant, resuspend the precipitate with 1×PBS buffer and centrifuge 3 times under the same centrifugation conditions to wash the unlabeled AMVLNs and excess DiI dye. Finally, resuspend the precipitate with 100 μL PBS buffer and collect it in an EP tube. Use it immediately or store it in a -80°C refrigerator. Use it as soon as possible to avoid fluorescence quenching.

[0074] (3) Flow cytometry and fluorescence microscopy were used to detect the internalization and absorption of AMVLNs by RAW264.7:

[0075] Dil-AMVLNs were co-cultured with RAW264.7 at different concentrations (2.5 μg / mL, 25 μg / mL) for 2 h, 4 h, and 8 h. Flow cytometry and fluorescence microscopy were then used to detect the internalization and absorption of AMVLNs by RAW264.7. The results of fluorescence microscopy are shown in Figure 2. Figure 6 As described (25 μg / mL), the flow cytometry results are as follows Figure 7 As shown, the results showed that Dil-AMVLNs could be internalized by RAW264.7 and the degree of internalization increased with the extension of co-incubation time.

[0076] Example 4

[0077] In vitro anti-inflammatory assay of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs):

[0078] In order to verify the biological activity of AMVLNs, in vitro cell level studies were first conducted to explore the anti-inflammatory effect of AMVLNs on RAW264.7.

[0079] (1) CCK-8 method was used to detect the effect of AMVLNs on the proliferation of RAW264.7 cells: RAW264.7 cells were cultured at a cell density of 1×10 4 Pieces / cm 2 Inoculated in 96-well plates. Different concentrations of AMVLNs (0.25 ng / mL, 2.5 ng / mL, 2.5 μg / mL and 25 μg / mL) were co-cultured with RAW264.7 cells, and the absorbance values ​​of each group of cells were measured at 450 nm after 12 h, 24 h, 48 h and 72 h of culture. The results showed that the selected concentrations of AMVLNs had neither an inhibitory nor a proliferative effect on RAW264.7.

[0080] (2) qRT-PCR detection of the effect of AMVLNs on the anti-inflammatory effect of RAW264.7: Mouse mononuclear macrophage cell line (RAW264.7) was used to simulate the in vitro inflammatory model. 6 / ml RAW264.7 cells were inoculated into culture flasks, and the cells were cultured with high-glucose DMEM complete medium containing 10% fetal bovine serum. After adding 1% double-antibody (streptomycin and penicillin), the cells were cultured in a constant temperature of 5% CO2 and 37°C. The medium was changed every 2 days, and the cells were passaged when they reached 90% confluence. Macrophages were cultured at 1×10 6 / mL was inoculated into a six-well plate and cultured overnight.

[0081] Macrophages were randomly divided into a normal control group, a model control group (LPS group), and an AMVLNs administration group. The normal control group and the model control group were added with high-glucose DMEM complete medium, and the AMVLNs group was pretreated with different concentrations of AMVLNs (0.1μg / mL, 0.25μg / mL, 0.5μg / mL) for 24h. The complete culture medium was then aspirated, and except for the normal control group, it was washed with PBS and replaced with 1μg / mL LPS solution, placed in a 37℃ incubator containing 5% CO2 for 24h, and the supernatant of each group of cells was collected, centrifuged, and stored at -80℃ for later use. The cells were collected separately using TRIzol lysis buffer and placed in sterilized EP tubes, marked, and qRT-PCR was performed to detect the expression of inflammatory factor-related genes (IL-1β, IL-6, and COX-2).

[0082] The results are as follows Figure 8 As shown, the results showed that 0.5 μg / mL AMVLNs could significantly reduce the expression of IL-1β, IL-6 and COX-2 mRNA.

[0083] (3) Flow cytometry was used to detect the effect of AMVLNs on the anti-inflammatory effect of RAW264.7: The cell culture was the same as in (2).

[0084] Macrophages were randomly divided into a normal control group, a model control group (LPS group), and an AMVLNs administration group. The normal control group and the model control group were added with high-glucose DMEM complete medium, and the AMVLNs group was pretreated with different concentrations of AMVLNs (0.25ng / mL, 2.5ng / mL) for 24h. The complete culture medium was then aspirated, and except for the normal control group, it was washed with PBS and replaced with 1μg / mL LPS solution, and placed in a 37℃ incubator with 5% CO2 for 24h. The cell supernatant of each group was collected, centrifuged, and stored at -80℃ for later use. Using the reactive oxygen detection kit (Biyuntian S0033), DCFH-DA was diluted with serum-free culture medium at 1:1000 to a final concentration of 10μmol / mL. The cell culture medium was removed and an appropriate volume of diluted DCFH-DA was added. Incubate in a 37℃ cell culture incubator for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. The cells were collected and analyzed by flow cytometry.

[0085] The results are as follows Fig. 9 As shown in the figure, it can be seen that LPS successfully induced an inflammatory model, AMVLPs reduced the reactive oxygen species produced by LPS, and AMVLPs had a good anti-inflammatory effect at the cellular level.

[0086] (4) Flow cytometry was used to detect the effect of AMVLNs on the antioxidant activity of RAW264.7: The cell culture was the same as in (2).

[0087] Macrophages were randomly divided into normal control group, model control group (H2O2 group), and AMVLNs administration group. The normal control group and model control group were added with high-glucose DMEM complete medium, and the AMVLNs group was pretreated with different concentrations of AMVLNs (0.25ng / mL, 2.5ng / mL) for 24h. The complete culture medium was then aspirated, and except for the normal control group, it was washed with PBS and replaced with 0.5mM H2O2 solution, placed in a 37℃ incubator with 5% CO2 for 24h, and the cell supernatant of each group was collected, centrifuged, and stored at -80℃ for later use. Using the reactive oxygen detection kit, DCFH-DA was diluted with serum-free culture medium at 1:1000 to a final concentration of 10μmol / mL. The cell culture medium was removed and an appropriate volume of diluted DCFH-DA was added. Incubate in a 37℃ cell culture incubator for 20 minutes. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. After collecting the cells, they were detected by flow cytometry.

[0088] The results are as follows Fig.10 As shown in the figure, it can be seen that H2O2 successfully induced the antioxidant model, AMVLNs reduced the reactive oxygen species produced by H2O2, and AMVLNs had a good antioxidant effect at the cellular level.

[0089] Example 5

[0090] Intestinal targeting trial of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs):

[0091] In vivo targeting studies are a prerequisite for subsequent studies on the biological activity of AMVLNs in vivo. Therefore, the targeted distribution of AMVLNs in vivo was detected before verifying the in vivo effects and mechanisms of AMVLNs.

[0092] (1) Preparation of Dil-AMVLNs solution: Take an appropriate amount of AMVLNs and add 1×PBS buffer to 1 mL. After mixing, add 10 μL DiI dye and label at 37°C in the dark for 30 min. Centrifuge at 4°C, 135,000×g for 70 min, discard the supernatant, resuspend the precipitate with 1×PBS buffer and centrifuge 3 times under the same centrifugation conditions to wash the unlabeled AMVLNs and excess DiI dye. Finally, resuspend the precipitate with 100 μL PBS buffer and collect it in an EP tube. Use it immediately or store it in a -80°C refrigerator. Use it as soon as possible to avoid fluorescence quenching.

[0093] (2) Animal in vivo imaging: 100 μL DiI-AMVLNs solution was injected into female C57BL / 6 mice through the tail vein, and blank control (administered with 100 μL PBS) and positive control (administered with 100 μL DiI dye solution (DiI:PBS=1:200)) were set up. Fluorescence imaging was performed 12h, 24h and 48h after administration.

[0094] The imaging results of the heart, liver, spleen, lung, kidney, femur and colon tissues of each group of mice are shown in Figure 2. Fig.11 As shown in the figure (the arrow on the right indicates the individual imaging comparison of the spleen tissue of each group of mice at 48 hours), it can be seen from the figure that no fluorescence signal was detected in the organs of the PBS group mice at any time point, the fluorescence signal of the DiI group mice was mainly distributed in the liver and lungs, and no signal was detected in the heart, kidney and spleen, and the fluorescence signal of the DiI-AMVLNs group mice was distributed in the organs distributed in the DiI group, and strong fluorescence signals also appeared in the colon tissue and spleen tissue, and tended to strengthen with the extension of time.

[0095] Example 6

[0096] In vivo trial of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs) for the treatment of ulcerative colitis:

[0097] (1) Establishment and grouping of ulcerative colitis mouse model:

[0098] 48 8-week-old female C57BL / 6 mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. Mice were grouped in groups of 5 per cage with free access to food and water. The experiment began after 1 week of adaptive feeding. They were randomly divided into a normal control group, a 2.5% DSS group, a high-concentration AMVLNs group (2.5 μg / mL), and a low-concentration group (0.5 μg / mL), with 12 mice in each group. Dextran sulfate sodium DSS was dissolved in distilled water to prepare a DSS solution with a concentration of 2.5%. The normal control group drank purified water freely, and the other groups drank 2.5% DSS solution freely for 5 days, 5 mL / day / mouse. Then the high and low concentration AMVLNs drug groups were gavaged for 5 days.

[0099] (2) Sample acquisition:

[0100] 24 hours after the intragastric administration, the mice were anesthetized with sodium pentobarbital to obtain samples, and the colon about 1 cm above the rectum was taken for tissue sections. The length of the colon of each group of mice was measured and photographed. The results are shown in Fig.12 and Fig.13 As shown in the figure, the colon length of the DSS model group was significantly shortened, and AMVLNs could restore the colon length.

[0101] The weight, stool characteristics and bloody stool of the mice were recorded every day. The weight changes of the mice in each group were as follows: Fig.14 As shown in the figure, it can be seen that the body weight of the DSS group decreased significantly, and AMVLNs could restore the body weight after DSS administration.

[0102] (3) Assessment of Disease Activity Index (DAI):

[0103] The disease activity index (DAI) is a comprehensive score of mouse weight, stool characteristics, and bleeding conditions, and is used to assess the severity of ulcerative colitis. According to a scoring system of 0-4, the weight, stool characteristics, and occult blood of the animals were observed daily, and the scores of each item were added together to obtain the DAI of each animal to assess the disease activity, with the highest score being 12 points. Observe and record the weight, stool characteristics, and bleeding of the mice daily. Formed stools are normal stools, pasty stools that do not adhere to the anus are loose stools, and those that adhere to the anus are watery stools. DAI score evaluation criteria: 0 points: no weight loss, normal stools, and no occult blood in the stool; 1 point: 1-5% weight loss, normal stools, and no occult blood in the stool; 2 points: 5-10% weight loss, semi-loose stools, and positive stools for occult blood; 3 points: 10-15% weight loss, semi-loose stools, and positive stools for occult blood; 4 points: weight loss is greater than or equal to 15%, loose stools, and blood in the stool is visible to the naked eye. The results are as follows: Fig.15 As shown, the results showed that the disease activity index in the AMVLNs high concentration group was significantly lower than that in the DSS group.

[0104] (4) HE staining to observe colon tissue:

[0105] After killing the mice, the colon tissue was rinsed with phosphate buffered saline (PBS), fixed in 10% neutral formaldehyde solution, then dehydrated and embedded in paraffin. 5 μm thick sections were taken for HE staining, and paraffin sections were dewaxed before staining. The paraffin sections of colon tissue were dewaxed in xylene twice, each for 15 minutes, and then dewaxed in a solution of xylene: anhydrous ethanol with a ratio of 1:1 for 5 minutes, then dewaxed in anhydrous ethanol twice, each for 5 minutes, and then dewaxed in 95%, 85%, and 75% ethanol for 5 minutes, and finally dewaxed in distilled water for 5 minutes. After dewaxing, stain with hematoxylin solution for 20 minutes, soak in 1% hydrochloric acid alcohol for 5 seconds, wash with tap water for 10 seconds, immerse in saturated lithium carbonate aqueous solution, and the sections return to blue for 10 seconds. The cells were soaked in 70% ethanol for 5 min, 80% ethanol for 5 min, 1% eosin alcohol solution for 1 min, anhydrous ethanol twice for 5 min each, anhydrous ethanol:xylene solution of 1:1 for 5 min, and xylene twice for 5 min each. The staining was completed, and the slides were sealed with neutral resin. The pathological changes of colon tissue were observed under a biological optical microscope. The HE staining results are shown in Fig.16 The results showed that the DSS group had obvious crypt gland destruction and atrophy, and the high-concentration group had a better therapeutic effect. The above results all show that AMVLNs can treat ulcerative colitis, and the high-concentration group has a better effect.

[0106] The above results show that the ulcerative colitis mouse model was successfully constructed and AMVLNs have the effect of treating ulcerative colitis.

[0107] Example 7

[0108] A trial of Atractylodes macrocephala-derived extracellular vesicles (AMVLNs) in preventing ulcerative colitis in vivo:

[0109] (1) Establishment and grouping of ulcerative colitis mouse model:

[0110] Forty-eight 8-week-old female C57BL / 6 mice were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. Mice were grouped in groups of 5 per cage with free access to food and water. The experiment began after 1 week of adaptive feeding. The mice were randomly divided into a normal control group, a 2.5% DSS group, a high-concentration AMVLNs group (2.5 μg / mL), and a low-concentration group (0.5 μg / mL), with 12 mice in each group. Dextran sulfate sodium DSS was dissolved in distilled water to prepare a 2.5% DSS solution. The normal control group was free to drink purified water, and the high and low concentration AMVLNs drug groups were first gavaged for 7 consecutive days, 5 mL / day / mouse. After the gavage, the other groups except the normal control group were free to drink 2.5% DSS solution for 5 days.

[0111] (2) Sample acquisition:

[0112] 24 hours after the intragastric administration, the mice were anesthetized with sodium pentobarbital to obtain samples, and the colon about 1 cm above the rectum was taken for tissue sections. The length of the colon of each group of mice was measured and photographed. The results are shown in Fig.17 and Fig.18 As shown in the figure, the colon length of the DSS model group was significantly shortened, and AMVLNs could prevent the change in colon length.

[0113] The weight, stool characteristics and bloody stool of the mice were recorded every day. The weight changes of the mice in each group were as follows: Fig.19 As shown in the figure, the body weight of the DSS group decreased significantly, and AMVLNs could prevent the body weight loss after DSS administration.

[0114] (3) Assessment of Disease Activity Index (DAI):

[0115] The disease activity index (DAI) is a comprehensive score of mouse weight, stool characteristics, and bleeding conditions, and is used to assess the severity of ulcerative colitis. According to a scoring system of 0-4, the weight, stool characteristics, and occult blood of the animals were observed daily, and the scores of each item were added together to obtain the DAI of each animal to assess the disease activity, with the highest score being 12 points. Observe and record the weight, stool characteristics, and bleeding of the mice daily. Formed stools are normal stools, pasty stools that do not adhere to the anus are loose stools, and those that adhere to the anus are watery stools. DAI score evaluation criteria: 0 points: no weight loss, normal stools, and no occult blood in the stool; 1 point: 1-5% weight loss, normal stools, and no occult blood in the stool; 2 points: 5-10% weight loss, semi-loose stools, and positive stools for occult blood; 3 points: 10-15% weight loss, semi-loose stools, and positive stools for occult blood; 4 points: weight loss is greater than or equal to 15%, loose stools, and blood in the stool is visible to the naked eye. The results are as follows: Fig. 20 As shown, the results showed that the disease activity index in the AMVLNs high concentration group was significantly lower than that in the DSS group.

[0116] (4) HE staining to observe colon tissue:

[0117] After killing the mice, the colon tissue was rinsed with phosphate buffered saline (PBS), fixed in 10% neutral formaldehyde solution, then dehydrated and embedded in paraffin. 5 μm thick sections were taken for HE staining, and paraffin sections were dewaxed before staining. The paraffin sections of colon tissue were dewaxed in xylene twice, each for 15 minutes, and then dewaxed in a solution of xylene: anhydrous ethanol with a ratio of 1:1 for 5 minutes, then dewaxed in anhydrous ethanol twice, each for 5 minutes, and then dewaxed in 95%, 85%, and 75% ethanol for 5 minutes, and finally dewaxed in distilled water for 5 minutes. After dewaxing, stain with hematoxylin solution for 20 minutes, soak in 1% hydrochloric acid alcohol for 5 seconds, wash with tap water for 10 seconds, immerse in saturated lithium carbonate aqueous solution, and the sections return to blue for 10 seconds. The cells were soaked in 70% ethanol for 5 min, 80% ethanol for 5 min, 1% eosin alcohol solution for 1 min, anhydrous ethanol twice for 5 min each, anhydrous ethanol:xylene solution of 1:1 for 5 min, and xylene twice for 5 min each. The staining was completed, and the slides were sealed with neutral resin. The pathological changes of colon tissue were observed under a biological optical microscope. The HE staining results are shown in Fig.21 The results showed that the DSS group had obvious crypt gland destruction and atrophy, and the high-concentration group had a better therapeutic effect. The above results all show that AMVLNs can prevent ulcerative colitis, and the high-concentration group has a better effect.

[0118] (5) qRT-PCR detection of the expression levels of inflammatory factors in mouse colon tissue:

[0119] Total RNA of tissues was extracted by Trizol method, and cDNA was synthesized by reverse transcription kit. Real-time quantitative PCR reaction was performed to detect mRNA of IL-10, IL-6, IL-1β, IL-12, and TNF-α. GAPDH was used as internal reference, and 2-ΔΔCt was used to analyze the relative expression of mRNA. The results are shown in Fig. 22 As shown, the results showed that AMVLNs could significantly reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors.

[0120] The above results show that the ulcerative colitis mouse model was successfully constructed and AMVLNs have the effect of preventing ulcerative colitis.

[0121] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, 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 various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0122] 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.

[0123] 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. Use of extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala in the preparation of a drug for preventing or treating ulcerative colitis; the extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala are prepared by an extraction method comprising the following steps: (1) filtering the juice of Atractylodes macrocephala raw material, centrifuging the filtrate and collecting the supernatant; (2) filtering the supernatant, performing ultracentrifugation, collecting the precipitate and resuspending the precipitate to obtain the obtained drug; The centrifugation and collection of the supernatant in step (1) are performed four times in total, the first centrifugation speed is 400-600g, the time is 5-15min; the second centrifugation speed is 1000-3000g, the time is 15-25min; the third centrifugation speed is 5000-7000g, the time is 20-40min; the fourth centrifugation speed is 9000-12000g, the time is 50-70min; The ultracentrifugation speed in step (2) is 90000-150000g, and the time is 50-100min.

2. The use according to claim 1, characterized in that: The resuspension was followed by filtration using a 0.22 μm filter.

3. The use according to claim 1, characterized in that: The dosage form of the drug is any one of dispersible tablets, sustained-release tablets, effervescent tablets, enteric-coated tablets, sustained-release capsules, enteric-coated capsules, granules, dripping pills or solutions.

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

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

6. Use of the extracellular vesicle-like nanoparticles derived from Atractylodes macrocephala as claimed in claim 1 in the preparation of colon-targeted preparations.

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